
Isofraxidin attenuates IL-1β-induced inflammatory response in human nucleus pulposus cells.
Su Xiaoqiang,Liu Bo,Gong Futai,Yin Jichao,Sun Qing,Gao Ye,Lv Zeyi,Wang Xiangyang
Journal of cellular biochemistry
Inflammation has been demonstrated to be the key factor for intervertebral disc degeneration (IVD), which remains a major public health problem. Isofraxidin is a coumarin compound that possesses strong anti-inflammatory activity. However, the role of isofraxidin in IVD remains unclear. The aim of this study was to evaluate the effects of isofraxidin on inflammatory response in human nucleus pulposus cells (NPCs) exposed to interleukin-1β (IL-1β). The results proved that isofraxidin attenuated the IL-1β-induced significant increases in inflammatory mediators and cytokines including nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), and IL-6. Besides, isofraxidin also inhibited the induction effect of IL-1β on matrix metalloproteinases (MMP)-3 and MMP-13. Moreover, the NF-κB activation caused by IL-1β was significantly inhibited by isofraxidin treatment. These findings suggested that isofraxidin alleviates IL-1β-induced inflammation in NPCs. Our work provided an idea that isofraxidin might act as a novel preventive role in IVD.
10.1002/jcb.28604
High-dose TGF-β1 degrades human nucleus pulposus cells via ALK1-Smad1/5/8 activation.
Qu Zhiqiang,Zhang Fengxiang,Chen Weiwei,Lin Tao,Sun Yongming
Experimental and therapeutic medicine
Transforming growth factor β1 (TGF-β1) can promote the proliferation and differentiation of intervertebral disc cells and participates in its repair process. However, whether TGF-β1 engages in the process of disc degeneration has not yet been fully elucidated. The present study aimed to investigate the function of high-dose TGF-β1 on the metabolism of nucleus pulposus cells (NPCs). TGF-β1 levels in human degenerative intervertebral disc tissues and tumor necrosis factor (TNF)-α-induced degenerative NPCs were analyzed. Furthermore, NPCs were treated with TGF-β1 and inhibitors of TGF-β1 receptors [ALK tyrosine kinase receptor (ALK) 1 and ALK5] to determine the effect of the receptors in the mediation of NPC degeneration. The NPC state was determined by the components of secretory collagen I/II, tissue inhibitor of metalloproteinase-3 (TIMP-3) and matrix metalloproteinase (MMP)-13. The mRNA expression of Smad1/2/3/5/8, the downstream gene of TGF-β1 mediated by ALK, was also measured. Results showed that TGF-β1 and ALK1 were positively associated with the degree of degeneration of NP or NPCs , but negatively associated with ALK5. Furthermore, high-doses of TGF-β1 suppressed collagen II, but enhanced collagen I, TIMP-3, MMP-13, ALK1/5 and Smad1/2/3/5/8 expression. ALK5 inhibition induced the suppression of Smad2/3 and aggravated high-dose TGF-β1-induced NPC degeneration, as shown by the reduction in collagen II and increase in collagen I, TIMP-3 and MMP-13. By contrast, ALK1 inhibition resulted in Smad1/5/8 suppression and alleviated high-dose TGF-β1-induced NPC degeneration. Taken together, it was concluded that high-doses of TGF-β1 contributed to the degeneration of NPCs via the upregulation of ALK1 and Smad1/5/8.
10.3892/etm.2020.9088
Inverse Agonist of Retinoid-Related Orphan Receptor-Alpha Prevents Apoptosis and Degeneration in Nucleus Pulposus Cells via Upregulation of YAP.
Mediators of inflammation
Intervertebral disc degenerative disease (IDD) is the most common degenerative spine disease, which leads to chronic low back pain and symptoms in the lower extremities. In this study, we found that ROR, a member of the retinoid-related orphan receptor family, is significantly elevated in nucleus pulposus tissue in IDD patients. The elevation of ROR is associated with increased apoptosis of nucleus pulposus (NP) cells. Therefore, we applicated a well-established inverse agonist of ROR, SR3335, to investigate its role in regulating NP cell metabolism and apoptosis. To further investigate the mechanism that SR3335 regulates the pathogenesis of IDD , tumor necrosis factor alpha (TNF-) stimulation was used in human NP cells to mimic the hostile environment that leads to degeneration. We found that SR3335 treatment reversed the trend of increased apoptosis in NP cells induced by TNF- treatment. Next, TNF- treatment upregulated the expression of type II collagen and aggrecan and downregulated MMP13 (matrix-degrading enzyme matrix metalloproteinase 13) and ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4). However, these effects were reversed after SR3335 treatment. Furthermore, we find that SR3335 mediated the effect in NP cells by regulating the YAP signaling pathway, especially by affecting the phosphorylation state of YAP. In conclusion, the reduction of matrix degradation enzymes and apoptosis upon SR3335 treatment suggests that SR3335 is a promising drug in reversing the deleterious microenvironment in IDD patients.
10.1155/2021/9954909
Oxygen metabolism-balanced engineered hydrogel microspheres promote the regeneration of the nucleus pulposus by inhibiting acid-sensitive complexes.
Bioactive materials
Intervertebral disc degeneration (IVDD) is commonly caused by imbalanced oxygen metabolism-triggered inflammation. Overcoming the shortcomings of antioxidants in IVDD treatment, including instability and the lack of targeting, remains challenging. Microfluidic and surface modification technologies were combined to graft chitosan nanoparticles encapsulated with strong reductive black phosphorus quantum dots (BPQDs) onto GelMA microspheres via amide bonds to construct oxygen metabolism-balanced engineered hydrogel microspheres (GM@CS-BP), which attenuate extracellular acidosis in nucleus pulposus (NP), block the inflammatory cascade, reduce matrix metalloproteinase expression (MMP), and remodel the extracellular matrix (ECM) in intervertebral discs (IVDs). The GM@CS-BP microspheres reduce HO intensity by 229%. Chemical grafting and electrostatic attraction increase the encapsulation rate of BPQDs by 167% and maintain stable release for 21 days, demonstrating the antioxidant properties and sustained modulation of the BPQDs. After the GM@CS-BP treatment, western blotting revealed decreased acid-sensitive ion channel-3 and inflammatory factors. Histological staining in an 8-week IVDD model confirmed the regeneration of NP. GM@CS-BP microspheres therefore maintain a balance between ECM synthesis and degradation by regulating the positive feedback between imbalanced oxygen metabolism in IVDs and inflammation. This study provides an in-depth interpretation of the mechanisms underlying the antioxidation of BPQDs and a new approach for IVDD treatment.
10.1016/j.bioactmat.2022.12.025
[Role of proteoglycans and glycosaminoglycans in the intervertebral disc degeneration].
Fortuniak Jan,Jaskólski Dariusz,Tybor Krzysztof,Komuński Piotr,Zawirski Marek
Neurologia i neurochirurgia polska
Proteoglycans and glycosaminoglycans are the main components of the extracellular matrix of the nucleus pulposus. Intervertebral disc degeneration due to aging and excessive mechanical loads results in adverse quantitative and structural changes to the macromolecules. Metalloproteinases induced by inflammatory mediators play a key role in degrading proteoglycans. Progressive matrix breakdown decreases water content in the disc. Dehydration compromises disc cells function and impairs resistance to compression. Biochemical changes may result in disc prolapse. Modifying the metabolism of proteoglycans and glycosaminoglycans might be an effective therapeutic strategy.
Expression of matrix metalloproteinase-1 (MMP-1) in Wistar rat's intervertebral disc after experimentally induced scoliotic deformity.
Grivas Theodoros B,Vasiliadis Elias S,Kaspiris Angelos,Khaldi Lubna,Kletsas Dimitris
Scoliosis
INTRODUCTION:A scoliotic deformity on intervertebral discs may accelerate degeneration at a molecular level with the production of metalloproteinases (MMPs). In the present experimental study we evaluated the presence of MMP-1 immunohistochemically after application of asymmetric forces in a rat intervertebral disc and the impact of the degree of the deformity on MMP-1 expression. MATERIAL-METHOD:Thirty female Wistar rats (aged 2 months old, weighted 200 ± 10 grams) were used. All animals were age, weight and height matched. A mini Ilizarov external fixator was applied at the base of a rat tail under anaesthesia in order to create a scoliotic deformity of the intervertebral disc between the 9th and 10th vertebrae. Rats were divided into three groups according to the degree of the deformity. In group I, the deformity was 10°, in group II 30° and in group III 50°. The rats were killed 35 days after surgery. The discs were removed along with the neighbouring vertebral bodies, prepared histologically and stained immunohistochemically. Immunopositivity of disc's cells for MMP-1 was determined using a semi-quantitative scored system. RESULTS:MMP-1 immunopositivity was detected in disc cells of annulus fibrosus of all intervertebral disc specimens examined. The percentage of MMP-1 positive disc cells in annulus fibrosus in group I, II and III were 20%, 43% and 75%, respectively. MMP-1 positivity was significantly correlated with the degree of the deformity (p < 0,001). An increase of chondrocyte-like disc cells was observed in the outer annulus fibrosus and at the margin of the intervertebral disc adjacent to the vertebral end plates. The difference in the proportion of MMP-1 positive disc cells between the convex and the concave side was statistically not significant in group I (p = 0,6), in group II this difference was statistically significant (p < 0,01). In group III the concave side showed a remarkable reduction in the number of disc's cells and a severe degeneration of matrix microstructure. CONCLUSION:The present study showed that an experimentally induced scoliotic deformity on a rat tail intervertebral disc results in over-expression of MMP-1, which is dependent on the degree of the deformity and follows a dissimilar distribution between the convex and the concave side.
10.1186/1748-7161-6-9
Overexpression of long non-coding RNA XIST promotes IL-1β-induced degeneration of nucleus pulposus cells through targeting miR-499a-5p.
He Jun,Yang Jing,Shen Tulan,He Jian
Molecular and cellular probes
BACKGROUND:Long non-coding RNA X-interactive specific transcript (XIST) is implicated in many diseases. However, its role and interaction with microRNA (miR)-499a-5p in intervertebral disc degeneration (IDD) remained unclear. METHODS:Nucleus pulposus (NP) tissue samples were collected and nucleus pulposus cells (NPCs) were isolated for Interleukin-1β (IL-1β) treatment and identification. XIST and miR-499a-5p expressions in the tissue were measured with quantitative real-time polymerase chain reaction (qRT-PCR). After IL-1β treatment, NPC apoptosis was detected by flow cytometry. The potential binding sites of XIST and miR-499a-5p were predicted by starBase and confirmed by dual-luciferase reporter assay. Relative expressions of tissue inhibitor of metalloproteinases-3 (TIMP-3), Matrix metalloproteinases-3 (MMP-3), MMP-13, Collagen II, Aggrecan and apoptosis-related proteins (Bcl-2 associated X protein, Bax; B-cell lymphoma 2, Bcl-2; cleaved caspase-3) were measured by qRT-PCR and Western blot as needed. RESULTS:XIST expression was upregulated in the NP tissues of patients with IDD, and IL-1β treatment resulted in a degradation of NPCs. Overexpressed XIST promoted the effects of IL-1β on increasing NPC apoptosis and expressions of XIST, MMP-3, MMP-13, Bax and Cleaved caspase-3, but down-regulated TIMP-3, Collagen II, Aggrecan and Bcl-2 expressions. Silencing XIST, however, showed the opposite effects to its overexpression. MiR-499a-5p expression was downregulated in NP tissues of IDD patients and could bind with XIST, while its upregulation reversed the effects of overexpressed XIST in the IL-1β-treated NPCs. CONCLUSION:Overexpressed XIST caused NPC degeneration through promoting apoptosis and extracellular matrix degradation of IL-1β-treated NPCs through targeting miR-499a-5p, and therefore can serve as a potential treatment for IDD.
10.1016/j.mcp.2021.101711
LincRNA-SLC20A1 (SLC20A1) promotes extracellular matrix degradation in nucleus pulposus cells in human intervertebral disc degeneration by targeting the miR-31-5p/MMP3 axis.
Yang Yang,Zhong Zhou,Zhao Yu,Ren Kuiyu,Li Ning
International journal of clinical and experimental pathology
Long non-coding RNAs (lncRNAs) are novel players in intervertebral disc degeneration (IDD) and show multiple functions. LncRNA lincRNA-SLC20A1 (SLC20A1) is aberrantly expressed in IDD. However, the role of SLC20A1 in degenerative nucleus pulposus (NP) cells and its underlying mechanism are unclear. The expressions of SLC20A1, miR-31-5p, and MMP3 were determined using RT-qPCR and western blotting. Extracellular matrix (ECM) degradation was evaluated by ECM-related genes collagen II, aggrecan, and ADAMTS4 using western blotting and an enzyme-linked immunosorbent assay (ELISA). The target binding between miR-31-5p and SLC20A1 or matrix metalloproteinase (MMP3) was predicted based on the or websites and confirmed using a luciferase reporter assay and an RNA pull-down assay. SLC20A1 expression is upregulated in NP tissues from IDD patients, and this expression promotes ADAMTS5 expression and represses collagen II and aggrecan expression in degenerative NP cells derived from IDD patients. Mechanically, SLC20A1 acts as a competing endogenous RNA (ceRNA) to negatively regulate miRNA-31-5p (miR-31-5p) expression. Moreover, MMP3 is a downstream target for miR-31-5p and is positively modulated by SLC20A1 in degenerative NP cells. Similar to the SLC20A1 effect in human NP cells, the downregulation of miR-31-5p facilitates ECM degradation as well. On the contrary, miR-31-5p upregulation abolishes the promoting role of SLC20A1 in degenerative NP cells, the effect of which is then blocked by the ectopic expression of MMP3. The upregulation of SLC20A1 aggravates ECM degradation in degenerative human NP cells by targeting the miR-31-5p/MMP3 axis, suggesting that the SLC20A1/miR-31-5p/MMP3 pathway can contribute to IDD progression.
Leptin Induces MMP-1 Expression Through the RhoA/ERK1/2/NF-κB Axis in Human Intervertebral Disc Cartilage Endplate-Derived Stem Cells.
Journal of inflammation research
Purpose:Intervertebral disc (IVD) degeneration, associated with aging, may cause low back pain and disability, with obesity as a significant risk factor. In a prior study, we found a positive correlation between IVD degeneration and levels of matrix metalloproteinase-1 (MMP-1) and leptin. Yet, the interaction between MMP-1 and leptin in IVD degeneration is unclear. Our research seeks to explore leptin's influence on MMP-1 expression and the underlying mechanisms in human intervertebral disc cartilage endplate-derived stem cells, specifically SV40 cells. Methods:The mRNA and protein expression in leptin-stimulated SV40 cells were assessed using RT-real-time PCR and Western blotting or ELISA, respectively. We examined leptin-mediated RhoA activation through a GTP-bound RhoA pull-down assay. Furthermore, the phosphorylation levels of mitogen-activated protein kinases and AKT in leptin-stimulated SV40 cells were analyzed using Western blotting. The activation of NF-κB by leptin was investigated by assessing phosphorylation of IKKα/β, IκBα, and NF-κB p65, along with the nuclear translocation of NF-κB p65. To understand the underlying mechanism behind leptin-mediated MMP-1 expression, we employed specific inhibitors. Results:Leptin triggered the mRNA and protein expression of MMP-1 in SV40 cells. In-depth mechanistic investigations uncovered that leptin heightened RhoA activity, promoted ERK1/2 phosphorylation, and increased NF-κB activity. However, leptin did not induce phosphorylation of JNK1/2, p38, or AKT. When we inhibited RhoA, ERK1/2, and NF-κB, it resulted in a decrease in MMP-1 expression. Conversely, inhibition of reactive oxygen species and NADPH oxidase did not yield the same outcome. Additionally, inhibiting RhoA or ERK1/2 led to a reduction in leptin-induced NF-κB activation. Moreover, inhibiting RhoA also decreased leptin-mediated ERK1/2 phosphorylation. Conclusion:These results indicated that leptin induced MMP-1 expression in SV40 cells through the RhoA/ERK1/2/NF-κB axis. This study provided the pathogenic role of leptin and suggested the potential therapeutic target for IVD degeneration.
10.2147/JIR.S431026
Microfluidic Chip with Low Constant-Current Stimulation (LCCS) Platform: Human Nucleus Pulposus Degeneration In Vitro Model for Symptomatic Intervertebral Disc.
Micromachines
Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) in the lumbar spine. This phenomenon is caused by several processes, including matrix degradation in IVD tissues, which is mediated by matrix metalloproteinases (MMPs) and inflammatory responses, which can be mediated by interactions among immune cells, such as macrophages and IVD cells. In particular, interleukin (IL)-1 beta (β), which is a master regulator secreted by macrophages, mediates the inflammatory response in nucleus pulposus cells (NP) and plays a significant role in the development or progression of diseases. In this study, we developed a custom electrical stimulation (ES) platform that can apply low-constant-current stimulation (LCCS) signals to microfluidic chips. Using this platform, we examined the effects of LCCS on IL-1β-mediated inflammatory NP cells, administered at various currents (5, 10, 20, 50, and 100 μA at 200 Hz). Our results showed that the inflammatory response, induced by IL-1β in human NP cells, was successfully established. Furthermore, 5, 10, 20, and 100 μA LCCS positively modulated inflamed human NP cells' morphological phenotype and kinetic properties. LCCS could affect the treatment of degenerative diseases, revealing the applicability of the LCCS platform for basic research of electroceuticals.
10.3390/mi12111291
Urolithin A Inhibits the Catabolic Effect of TNFα on Nucleus Pulposus Cell and Alleviates Intervertebral Disc Degeneration .
Liu Huiyong,Kang Honglei,Song Chao,Lei Zuowei,Li Li,Guo Jianfeng,Xu Yong,Guan Hanfeng,Fang Zhong,Li Feng
Frontiers in pharmacology
Low back pain (LBP) is a common worldwide disease that causes an enormous social economic burden. Intervertebral disc degeneration (IDD) is considered as a major cause of LBP. The process of IDD is complicated and involves both inflammation and senescence. The production of pro-inflammatory cytokines, including tumor necrosis factor (TNF)α and interleukin (IL)-1β, is increased in the degenerating intervertebral disc, inducing extracellular matrix degradation. Urolithin A (UA) is a metabolite compound resulting from the degradation of ellagitannins by gut bacteria. UA has been reported to be useful for the treatment of diseases associated with inflammation, senescence, and oxidative damage. Therefore, we hypothesized that UA may be an effective treatment for IDD. This study examined the effects of UA on IDD and and explored their underlying mechanisms. Our findings indicated that UA could attenuate cellular senescence induced by hydrogen peroxide in nucleus pulposus cells. UA treatment decreased TNFα-induced matrix metalloproteinase production and the loss of collagen II. At the molecular level, UA considerably blocked the phosphorylation of the extracellular signal-regulated kinase, c-JUN N-terminal kinase, and Akt pathways. study illustrated that UA treatment could ameliorate IDD in a needle-punctured rat tail model, which was evaluated by X-ray imaging, magnetic resonance imaging, and histological analysis. Thus, the results of our study revealed that UA may be a useful therapeutic agent for the treatment of IDD.
10.3389/fphar.2018.01043
Gallic acid inhibits the release of ADAMTS4 in nucleus pulposus cells by inhibiting p65 phosphorylation and acetylation of the NF-κB signaling pathway.
Huang Yao,Chen Jian,Jiang Tao,Zhou Zheng,Lv Bin,Yin Guoyong,Fan Jin
Oncotarget
This study investigated the inhibitory effect of gallic acid (GA) on the release of A Disintegrin and Metalloproteinase with Thrombospondin motifs 4 (ADAMTS4) through the regulation of the NF-κB signaling pathway, which is closely related to the matrix metalloproteinases in nucleus pulposus cells. Different concentrations of GA were added to TNF-α-induced human nucleus pulposus cells (hNPCs) and intervertebral disc degeneration rat model. ADAMTS-4 expression increased both in the TNF-α-induced nucleus pulposus cells and intervertebral disc degeneration rat model. By contrast, the release of ADAMTS-4 was reduced, and the TNF-α-induced apoptosis of nucleus pulposus cells was significantly inhibited after addition of GA at different concentrations. Further study found that the levels of phosphorylated p65 (p-p65) was increased and the classical NF-κB signal pathway was activated after the nucleus pulposus cells were stimulated by TNF-α. Meanwhile, GA suppressed the p65 phosphorylation and inceased p65 deacetylation levels. As a consequence, GA can decrease the expression of ADAMTS-4 in nucleus pulposus cells by regulating the phosphorylation and acetylation of p65 in NF-κB signaling pathways.
10.18632/oncotarget.17509
The aging spine: the role of inflammatory mediators in intervertebral disc degeneration.
Podichetty Vinod K
Cellular and molecular biology (Noisy-le-Grand, France)
Human intervertebral disc undergoes multifactorial biochemical and morphologic degenerative changes during the process of aging. The frequency of degeneration, especially lumbar degeneration increases sharply with age and is regarded as a major cause of discogenic low back pain. Since degenerative discs are often asymptomatic, the pathobiology of discogenic back pain remains unclear. Degenerated discs spontaneously produce increased amounts of inflammatory mediators suggesting their role in the degenerative process of the intervertebral disc. However, the relationship between aging, degenerative processes, and actual illness is far from clear. Basic science research has demonstrated that the intervertebral disc is an avascular tissue element occupied by inadequately characterized cells in an extensive extracellular matrix. While the annulus fibrosus is predominantly collagenous, the matrix of the central nucleus pulposus is rich in proteoglycans. With aging, the substance of proteoglycans significantly decreases which is believed to be a critical factor in intervertebral disc degeneration. A variety of inflammatory mediators have been implicated in the degeneration of the intervertebral disc including nitric oxide (NO), interleukins, matrix metalloproteinases (MMP), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-alpha) and a group of cytokines. MMPs, PGE2, and a variety of cytokines have been already been shown to play a role in the degradation of articular cartilage. Nitric oxide is a novel mediator that is drawn into much attention recently for its role in disc abnormalities. Elevated nitric oxide production derived from NO synthase activity has been manifested in cerebrospinal fluid in patients with degenerative lumbar disease. However, the regulatory mechanism of NO and its relationship to the clinical manifestations are unclear. The biochemical events that occur with the 'aging spine' and in particular, the role of inflammatory mediators in intervertebral disc degeneration have not been studied assertively. Correspondingly, the association between degeneration of the intervertebral disc and the nociceptive mechanism of back pain is also not fully elucidated. However, there is high incidence of degenerated disc disorders manifested as back and neck pain and are among the most commonly encountered complaints in elderly population. It is hypothesized that the degenerative cascade ultimately leads to extensive structural defects and loss of normal motion segment function and configuration.
Mechanism of microRNA-146a-mediated IL-6/STAT3 signaling in lumbar intervertebral disc degeneration.
Experimental and therapeutic medicine
The aim of the study was to investigate the mechanism of microRNA (miR)-146a-mediated activation of interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) in lumbar intervertebral disc degeneration. To obtain intervertebral tissue, we recruited 5 patients with lumbar intervertebral disc herniation (experimental group) and 5 patients with lumbar burst fracture (control group). Nucleus pulposus tissue was extracted by surgery and cultured. miR-146a empty vector, mimic, and inhibitor were transfected into the two groups of cells for 24 h and the levels of IL-6, type II collagen (Col II), aggrecan, STAT3, matrix metalloproteinase (MMP)-3, and a disintegrin and metalloproteinase with thrombospondin type I motifs (ADAMTS) were detected. We found no differences in the levels of IL-6, Col II, aggrecan, STAT3, MMP-3, and ADAMTS before and after treatment in the control group. However, the levels of miR-146a, IL-6, STAT3, MMP-3, and ADAMTS were significantly elevated, whereas Col II and aggrecan levels were lower in the experimental group before treatment. The levels of IL-6, STAT3, MMP-3, and ADAMTS were elevated after treatment with miR-146a mimic when compared with the miR-146a empty vector in the experimental group, and Col II and aggrecan levels were decreased. However, the cells treated with miR-146a inhibitor had the opposite result. Thus, the IL-6/STAT3 signaling pathway can be mediated by a high expression of miR-146a to regulate the occurrence of lumbar intervertebral disc degeneration.
10.3892/etm.2017.4611
Matrix synthesis and degradation in human intervertebral disc degeneration.
Le Maitre C L,Pockert A,Buttle D J,Freemont A J,Hoyland J A
Biochemical Society transactions
Degeneration of the intervertebral disc has been implicated in chronic low back pain. Type II collagen and proteoglycan (predominantly aggrecan) content is crucial to proper disc function, particularly in the nucleus pulposus. In degeneration, synthesis of matrix molecules changes, leading to an increase in the synthesis of collagens type I and III and a decreased production of aggrecan. Linked to this is an increased expression of matrix-degrading molecules including MMPs (matrix metalloproteinases) and the aggrecanases, ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) 1, 4, 5, 9 and 15, all of which are produced by native disc cells. Importantly, we have found that there is a net increase in these molecules, over their natural inhibitors [TIMP-1 (tissue inhibitor of metalloproteinases-1), 2 and 3], suggesting a deregulation of the normal homoeostatic mechanism. Growth factors and cytokines [particularly TNFalpha (tumour necrosis factor alpha) and IL-1 (interleukin 1)] have been implicated in the regulation of this catabolic process. Our work has shown that in degenerate discs there is an increase in IL-1, but no corresponding increase in the inhibitor IL-1 receptor antagonist. Furthermore, treatment of human disc cells with IL-1 leads to a decrease in matrix gene expression and increased MMP and ADAMTS expression. Inhibition of IL-1 would therefore be an important therapeutic target for preventing/reversing disc degeneration.
10.1042/BST0350652
Resveratrol attenuated TNF-α-induced MMP-3 expression in human nucleus pulposus cells by activating autophagy via AMPK/SIRT1 signaling pathway.
Wang Xiao-Hu,Zhu Lei,Hong Xin,Wang Yun-Tao,Wang Feng,Bao Jun-Ping,Xie Xin-Hui,Liu Lei,Wu Xiao-Tao
Experimental biology and medicine (Maywood, N.J.)
Resveratrol (RSV) is known to play a role of anti-TNF-α in a number of cell types. However, whether RSV modulates the effects of TNF-α on human nucleus pulposus (NP) cells is unknown. The purpose of this study is to investigate whether RSV regulates TNF-α-induced matrix metalloproteinase-3 (MMP-3) expression. Via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we found that MMP-3 expression induced by TNF-α was inhibited by RSV treatment. Depending on Western blot and qRT-PCR assay, we found that RSV induced autophagy in human NP cells, whereas inhibition of autophagy remarkably abolished the restraining role of RSV in the TNF-α-mediated up-regulation of MMP-3. Furthermore, RSV increased SIRT1 expression and SIRT1 knockdown significantly suppressed RSV-induced autophagy in NP cells. RSV also activated AMP-activated protein kinase (AMPK), while inhibition of AMPK notably abolished RSV-induced SIRT1 expression. Our data showed that RSV attenuated TNF-α-induced MMP-3 expression in human NP cells by activating autophagy via AMPK/SIRT1 signaling pathway. This new finding suggested that RSV might act as a novel preventive and therapeutic role in intervertebral disc degeneration.
10.1177/1535370216637940
Genetic Predictors of Early-Onset Spinal Intervertebral Disc Degeneration: Part One of Two.
Fiani Brian,Covarrubias Claudia,Jarrah Ryan
Cureus
Intervertebral disc (IVD) degeneration is a progressive and painful pathology that can root from mechanical, biochemical, and environmental stressors. However, recent advancements in biogenetics have now found a predominating genetic influence. Nevertheless, despite these advancements, the pathophysiology of IVD degeneration remains poorly understood. In the first of our two-part series, we will characterize some of the most recent and best-studied genes in the context of intervertebral disc degeneration. We will attempt to formulate the first contemporary gene guide that characterizes the genetic profile of IVD degeneration. The genes of interest include aggrecan (ACAN), matrix metalloproteinase 2 (MMP2), vitamin D receptor (VDR), interleukin 1 alpha (IL1A), and those encoded for collagens such as collagen type XI alpha 1 chain (COL11A1), collagen type I alpha 1 chain (COL1A1), collagen type IX alpha 2 chain (COL9A2), and collagen type IX alpha 3 chain (COL9A3). Genetic analysis studies reveal that these genes play vital roles in maintaining the structural integrity of the intervertebral disc, activating enzymes involved in the extracellular matrix, and promoting connective tissue formation. Nevertheless, characterizing these genes alone is not enough to understand the pathophysiology of IVD degeneration. Therefore, further studies are warranted to understand molecular signalling pathways of IVD degeneration better and ultimately create more sophisticated genetic and cell-based therapies to improve patient outcomes.
10.7759/cureus.15182
Action of fibroblast growth factor-2 on the intervertebral disc.
Li Xin,An Howard S,Ellman Michael,Phillips Frank,Thonar Eugene J,Park Daniel K,Udayakumar Ranjith K,Im Hee-Jeong
Arthritis research & therapy
INTRODUCTION:Fibroblast growth factor 2 (FGF2) is a growth factor that is immediately released after cartilage injury and plays a pivotal role in cartilage homeostasis. In human adult articular cartilage, FGF2 mediates anti-anabolic and potentially catabolic effects via the suppression of proteoglycan (PG) production along with the upregulation of matrix-degrading enzyme activity. The aim of the present study was to determine the biological effects of FGF2 in spine disc cells and to elucidate the complex biochemical pathways utilized by FGF2 in bovine intervertebral disc (IVD) cells in an attempt to further understand the pathophysiologic processes involved in disc degeneration. METHODS:We studied the effect of FGF2 on IVD tissue homeostasis by assessing MMP-13 expression (potent matrix-degrading enzyme), PG accumulation, and PG synthesis in the bovine spine IVD, as well as evaluating whether FGF2 counteracts known anabolic factors such as BMP7. To understand the molecular mechanisms by which FGF2 antagonizes BMP7 activity, we also investigated the signaling pathways utilized by FGF2 in bovine disc tissue. RESULTS:The primary receptor expressed in bovine nucleus pulposus cartilage is FGFR1, and this receptor is upregulated in degenerative human IVD tissue compared with normal IVD tissue. Stimulation of bovine nucleus pulposus cells cultured in monolayer with FGF2 augmented the production of MMP-13 at the transcriptional and translational level in a dose-dependent manner. Stimulation of bovine nucleus pulposus cells cultured in alginate beads for 21 days with FGF2 resulted in a dose-dependent decrease in PG accumulation, due at least in part to the inhibition of PG synthesis. Further studies demonstrate that FGF2 (10 ng/ml) antagonizes BMP7-mediated acceleration of PG production in bovine nucleus pulposus cells via the upregulation of noggin, an inhibitor of the transforming growth factor beta/bone morphogenetic protein signaling pathway. Chemical inhibitor studies showed that FGF2 utilizes the mitogen-activated protein kinase and NF-kappaB pathways to upregulate noggin, serving as one potential mechanism for its anti-anabolic effects. CONCLUSION:FGF2 is anti-anabolic in bovine spine disc cells, revealing the potential of FGF2 antagonists as unique biologic treatments for both prevention and reversal of IVD degeneration.
10.1186/ar2407
Interleukin-2 is upregulated in patients with a prolapsed lumbar intervertebral disc and modulates cell proliferation, apoptosis and extracellular matrix metabolism of human nucleus pulposus cells.
Wang Zhirong,Wang Genlin,Zhu Xuesong,Geng Dechun,Yang Huilin
Experimental and therapeutic medicine
Previous studies have demonstrated that the expression levels of cytokines are increased in degenerated intervertebral disc tissues, and several cytokines are associated with the pathogenesis of intervertebral disc degeneration. However, the role of interleukin (IL)-2 in the cellular functions of intervertebral disc tissues remains unreported. The present study aimed to determine the expression levels of IL-2 in the nucleus pulposus (NP) tissues of patients with a prolapsed lumbar intervertebral disc; and to observe the changes in cell proliferation, apoptosis, extracellular matrix (ECM) metabolism and p38 mitogen-activated protein kinase (MAPK) signaling in human NP cells (HNPCs) following treatment with IL-2. The present study demonstrated that IL-2 expression levels were upregulated in the NP tissues of patients with a prolapsed lumbar intervertebral disc; and a subsequent MTT assay demonstrated that IL-2 inhibits the proliferation of HNPCs in a dose-dependent manner. Furthermore, as demonstrated by the increased protein expression levels of Fas cell surface death receptor and the induction of caspase-8 and caspase-3 activity, the death receptor pathway was activated by IL-2 in the HNPCs in order to promote cell apoptosis. In addition, IL-2 promoted ECM degradation in the HNPCs, as demonstrated by an increase in the expression levels of type I collagen, a disintegrin and metalloproteinase with thrombospondin motifs and matrix metalloproteinases, and decreased aggrecan and type II collagen expression levels. Furthermore, phosphorylated-p38 was significantly increased in the HNPCs following IL-2 treatment. In conclusion, the present study demonstrated that IL-2 inhibits cell proliferation, and induces cell apoptosis and ECM degradation, accompanied by the activation of p38 MAPK signaling in HNPCs. Therefore, IL-2 may be a potential therapeutic agent for the treatment of degenerative disc disease.
10.3892/etm.2015.2809
HtrA1 up-regulates expression of MMPs via Erk1/2/Rock-dependent pathways.
Li Dapeng,Yue Jiawei,Wu Yan,Barnie Prince Amoah,Wu Yumin
International journal of clinical and experimental pathology
BACKGROUND:There are few studies that have identified the potential role of a high temperature requirement A1 (HtrA1) in intervertebral disc degeneration (IDD). This study was undertaken to investigate the regulatory role of HtrA1 in the pathogenesis of IDD. MATERIAL AND METHODS:The mRNA levels of HtrA1 and matrix metalloproteinases (MMPs) of human intervertebral disc degeneration tissues were measured by real-time quantitative PCR, and a correlation between the expression level of HtrA1 and MMPs was also investigated. Human nucleus pulposus cells (HNPCs) were challenged with rHtrA1, and expression of MMPs was measured by real-time quantitative PCR, Western blotting, and ELISA. Moreover, to analyze the mechanism by which HtrA1 up-regulates MMPs, ERK1/2/ROCK signaling pathway inhibitors were also used. RESULTS:We found significant increases in mRNA expression of HtrA1 and MMP1, 3, 9, and 13 in IDD tissues compared with control. HtrA1 expression level was associated with the levels of MMP1, 3, and 13. Expression of MMP1, 3, and 13 mRNA and protein were significantly increased in HNPCs treated by rHtrA1. Moreover, administration of the ERK1/2 signaling pathway inhibitor or ROCK signaling pathway inhibitor decreased rHtrA1-induced MMPs production. Therefore, changes in HtrA1 expression could be involved in the pathogenesis of IDD. CONCLUSION:Our findings indicate that HtrA1 can induce increases in MMPs in HNPCs via the ERK1/2/ROCK signaling pathway, thus providing new insights into the role of HtrA1 in the pathogenesis of IDD.
Degenerative grade affects the responses of human nucleus pulposus cells to link-N, CTGF, and TGFβ3.
Abbott Rosalyn D,Purmessur Devina,Monsey Robert D,Brigstock David R,Laudier Damien M,Iatridis James C
Journal of spinal disorders & techniques
STUDY DESIGN:Cells isolated from moderately and severely degenerated human intervertebral disks (IVDs) cultured in an alginate scaffold. OBJECTIVE:To compare the regenerative potential of moderately versus severely degenerated cells using 3 proanabolic stimulants. SUMMARY OF BACKGROUND DATA:Injection of soluble cell signaling factors has potential to slow the progression of IVD degeneration. Although degenerative grade is thought to be an important factor in targeting therapeutic interventions it remains unknown whether cells in severely degenerated IVDs have impaired metabolic functions compared to lesser degenerative levels or if they are primarily influenced by the altered microenvironment. METHODS:Nucleus pulposus (NP) cells were cultured in alginate for 21 days and treated with 3 different proanabolic stimulants: a growth factor/anti-inflammatory combination of transforming growth factor β3 (TGFβ3)+dexamethasone (Dex), or matricellular proteins connective tissue growth factor (CTGF) or Link-N. They were assayed for metabolic activity, DNA content, glycosaminoglycan, and qRT-PCR gene profiling. RESULTS:Moderately degenerated cells responded to stimulation with increased proliferation, decreased IL-1β, MMP9, and COL1A1 expression, and upregulated HAS1 as compared with severely degenerated cells. TGFβR1 (ALK5) receptors were expressed at greater levels in moderately than severely degenerated cells. TGFβ3+Dex had a notable stimulatory effect on moderately degenerated NP cells with increased anabolic gene expression and decreased COL1A1 and ADAMTS5 gene expression. Link-N and CTGF had similar responses in all assays, and both treatments upregulated IL-1β expression and had a more catabolic response than TGFβ3+Dex, particularly in the more severely degenerated group. All groups, including different degenerative grades, produced similar amounts of glycosaminoglycan. CONCLUSIONS:Proanabolic stimulants alone had limited capacity to overcome the catabolic and proinflammatory cytokine expression of severely degenerated NP cells and likely require additional anti-inflammatory treatments. Moderately degenerated NP cells had greater TGFβ receptor 1 expression and better responded to anabolic stimulation.
10.1097/BSD.0b013e31826e0ca4
The involvement of interleukin-1 and interleukin-4 in the response of human annulus fibrosus cells to cyclic tensile strain: an altered mechanotransduction pathway with degeneration.
Gilbert Hamish T J,Hoyland Judith A,Freemont Anthony J,Millward-Sadler Sarah J
Arthritis research & therapy
INTRODUCTION:Recent evidence suggests that intervertebral disc (IVD) cells derived from degenerative tissue are unable to respond to physiologically relevant mechanical stimuli in the 'normal' anabolic manner, but instead respond by increasing matrix catabolism. Understanding the nature of the biological processes which allow disc cells to sense and respond to mechanical stimuli (a process termed 'mechanotransduction') is important to ascertain whether these signalling pathways differ with disease. The aim here was to investigate the involvement of interleukin (IL)-1 and IL-4 in the response of annulus fibrosus (AF) cells derived from nondegenerative and degenerative tissue to cyclic tensile strain to determine whether cytokine involvement differed with IVD degeneration. METHODS:AF cells were isolated from nondegenerative and degenerative human IVDs, expanded in monolayers and cyclically strained in the presence or absence of the cytokine inhibitors IL-1 receptor antagonist (IL-1Ra) or IL-4 receptor antibody (IL-4RAb) with 10% strain at 1.0 Hz for 20 minutes using a Flexcell strain device. Total RNA was extracted from the cells at time points of baseline control and 1 or 24 hours poststimulation. Quantitative real-time polymerase chain reaction was used to analyse the gene expression of matrix proteins (aggrecan and type I collagen) and enzymes (matrix metalloproteinase 3 (MMP3) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 4 (ADAMTS4)). RESULTS:Expression of catabolic genes (MMP3 and ADAMTS4) decreased in AF cells derived from nondegenerative tissue in response to 1.0-Hz stimulation, and this decrease in gene expression was inhibited or increased following pretreatment of cells with IL-1Ra or IL-4RAb respectively. Treatment of AF cells derived from degenerative tissue with an identical stimulus (1.0-Hz) resulted in reduced anabolic gene expression (aggrecan and type I collagen), with IL-1Ra or IL-4RAb pretreatment having no effect. CONCLUSIONS:Both IL-1 and IL-4 are involved in the response of AF cells derived from nondegenerative tissue to 1.0-Hz cyclic tensile strain. Interestingly, the altered response observed at 1.0-Hz in AF cells from degenerative tissue appears to be independent of either cytokine, suggesting an alternative mechanotransduction pathway in operation.
10.1186/ar3229
Duhuo Jisheng Decoction inhibits SDF-1-induced inflammation and matrix degradation in human degenerative nucleus pulposus cells in vitro through the CXCR4/NF-κB pathway.
Liu Zong-Chao,Wang Zhen-Long,Huang Chen-Yi,Fu Zhi-Jiang,Liu Yong,Wei Zhang-Chao,Liu Shi-Gui,Ma Chuan,Shen Jie-Liang,Duan Dayue Darrel
Acta pharmacologica Sinica
Lower back pain (LBP) is the most common disease in orthopedic clinics world-wide. A classic Fangji of traditional Chinese medicine, Duhuo Jisheng Decoction (DHJSD), has been proven clinically effective for LBP but its therapeutic mechanisms remain unclear. We hypothesized that DHJSD might relieve LBP through inhibiting the exaggerated proinflammatory cytokines and extracellular matrix (ECM) degradation. Thus, we studied the effects of DHJSD on stromal cell-derived factor-1 (SDF-1)-induced inflammation and ECM degradation in human nucleus pulposus cells (hNPCs). The primary hNPCs were isolated from either degenerated human intervertebral disc (HID) of LBP patients or normal HID of lumbar vertebral fracture patients, and cultured in vitro. The cells were treated with SDF-1 (10 ng/mL) and subsequently with different concentrations (100-500 μg/mL) of DHJSD for 24 h, respectively. We found that application of DHJSD significantly antagonized the SDF-1-induced production of proinflammatory cytokines and reduction of aggrecan and type II collagen in the hNPCs. DHJSD also markedly reduced the SDF-1-induced increase of CXCR4 and p-p65 and inhibited the nuclear translocation of p65 in the hNPCs. DHJSD, CXCR4-siRNA, and NF-κB inhibitor (BAY11-7082) caused the same inhibition of exaggerated proinflammatory cytokines in the SDF-1-treated hNPCs. These results provided compelling evidence that DHJSD may inhibit the generation of proinflammatory mediators and ECM degradation of HID through an orchestrated targeting at multiple molecules in the SDF-1/CXCR4/NF-κB pathway, thus offered novel mechanistic insights into the clinical effectiveness of DHJSD on LBP.
10.1038/aps.2018.36
Towards biological anulus repair: TGF-β3, FGF-2 and human serum support matrix formation by human anulus fibrosus cells.
Hegewald Aldemar A,Zouhair Sabra,Endres Michaela,Cabraja Mario,Woiciechowsky Christian,Thomé Claudius,Kaps Christian
Tissue & cell
Closure and biological repair of anulus fibrosus (AF) defects in intervertebral disc diseases is a therapeutic challenge. The aim of our study was to evaluate the anabolic properties of bioactive factors on cartilaginous matrix formation by AF cells. Human AF cells were harvested from degenerated lumbar AF tissue and expanded in monolayer culture. AF cell differentiation and matrix formation was initiated by forming pellet cultures and stimulation with hyaluronic acid (HA), human serum (HS), fibroblast growth factor-2 (FGF-2), transforming growth factor-β3 (TGF-β3) and TGF-β3/FGF-2 for up to 4 weeks. Matrix formation was assessed histologically by staining of proteoglycan, type I and type II collagens and by gene expression analysis of typical extracellular matrix molecules and of catabolic matrix metalloproteinases MMP-2 and MMP-13. AF cells, stimulated with HS, FGF-2 and most pronounced with TGF-β3 or TGF-β3/FGF-2 formed a cartilaginous matrix with significantly enhanced expression of matrix molecules and of MMP-13. Stimulation of AF cells with TGF-β3 was accompanied by induction of type X collagen, known to occur in hypertrophic cartilage cells having mineralizing potential. HA did not show any chondro-inductive characteristics. These findings suggest human serum, FGF-2 and TGF-β3 as possible candidates to support biological treatment strategies of AF defects.
10.1016/j.tice.2012.09.011
LIPUS inhibits inflammation and catabolism through the NF-κB pathway in human degenerative nucleus pulposus cells.
Yi Weiwei,Chen Qing,Liu Chuan,Li Kaiting,Tao Bailong,Tian Guihua,Zhou Lu,Li Xiaohong,Shen Jieliang,Liu Bo,Hu Zhenming,Wang Dawu,Bai Dingqun
Journal of orthopaedic surgery and research
BACKGROUND:Low-intensity pulsed ultrasound (LIPUS) is a safe and noninvasive rehabilitative physical therapy with anti-inflammatory effects. The current study investigated the effect of LIPUS on the inflammation of nucleus pulposus (NP) cells and its underlying mechanism. METHODS:Human NP cells were acquired from lumbar disc herniation tissue samples and cultured for experiments. Human NP cells were treated with LPS and then exposed to LIPUS (15 mW/cm, 30 mW/cm and 60 mW/cm) for 20 min daily for 3 days to determine the appropriate intensity to inhibit the expression of the inflammatory factors TNF-α and IL-1β. The gene and protein expression of aggrecan, collagen II, MMP-3 and MMP-9 was measured by real-time PCR and western blotting, respectively. The activity of the nuclear factor-kappa B (NF-κB) pathway was examined by western blotting and immunofluorescence. After pretreatment with the NF-κB inhibitor PDTC, the expression of TNF-α, IL-1β, MMP-3 and MMP-9 was measured by real-time PCR. RESULTS:LIPUS at intensities of 15 mW/cm, 30 mW/cm and 60 mW/cm inhibited LPS-induced NP cell expression of the inflammatory factors TNF-α and IL-1β, especially at 30 mW/cm. LIPUS significantly upregulated the gene and protein expression of aggrecan and collagen II and downregulated the gene and protein expression of MMP-3 and MMP-9 in LPS-induced NP cells. The NF-κB signaling pathway was inhibited by LIPUS through inhibiting the protein expression of p-P65 and the translocation of P65 into the nucleus in LPS-induced NP cells. In addition, LIPUS had similar effects as the NF-κB inhibitor PDTC by inhibiting the NF-κB signaling pathway, inflammation and catabolism in LPS-induced human degenerative nucleus pulposus cells. CONCLUSION:LIPUS inhibited inflammation and catabolism through the NF-κB pathway in human degenerative nucleus pulposus cells.
10.1186/s13018-021-02739-1
Human nucleus pulposus cells react to IL-6: independent actions and amplification of response to IL-1 and TNF-α.
Studer Rebecca K,Vo Nam,Sowa Gwendolyn,Ondeck Courtney,Kang James
Spine
STUDY DESIGN.: Human nucleus pulposus cells were activated with IL-6 plus IL-6 soluble receptor (sR) in the presence or absence of IL-1β or TNF-α. Cell production of factors modulating the anabolic/catabolic balance of the disc and proteoglycan synthesis were determined. OBJECTIVE.: To evaluate NP cell response to exogenous IL-6, and how IL-6 modulates IL-1 and TNF-α actions in these cells. SUMMARY OF BACKGROUND DATA.: Interleukin-6 (IL-6) is produced by cervical and lumbar herniated discs and is associated with neurological symptoms of intervertebral disc degeneration. It upregulates catabolic gene expression and downregulates matrix protein gene expression in chondrocytes. However, no studies have evaluated the effects of IL-6 on disc nucleus pulposus (NP) cells. METHODS.: NP cells from degenerated human discs were expanded in monolayer, maintained in alginate bead culture, and activated with IL-6 plus IL-6 soluble receptor (sR), in the presence or absence of IL-1β or TNF-α. Conditioned media was collected and analyzed for nitrite, PGE-2, TIMP-1, MMP-3, VEGF, and IL-8. Proteoglycan synthesis was assayed as S-sulfate incorporation normalized to DNA content and relative gene expression measured by rtPCR. RESULTS.: IL-6 + sR decreased collagen and aggrecan message, proteoglycan synthesis, and exacerbated the downregulation of proteoglycan synthesis effected by IL-1. PGE-2 synthesis was increased by IL-6 + sR, as was the induction of COX-2 mRNA. IL-6 + sR also enhanced IL-1 and TNF-α stimulated synthesis of PGE-2. IL-6 + sR induced MMP-3 approximately twofold and increased gene expression and synthesis in cells exposed to IL-1 and TNF-α. MMP-13 induction by TNF-α was also potentiated by IL-6 + sR. IL-6 + sR induced IL-6 gene expression and increased that stimulated by TNF-α fourfold. CONCLUSION.: The results suggest maneuvers to diminish IL-6 production in the disc could provide some protection against the adverse effects of IL-1 and TNF-α, thus, helping preserve disc composition, structure, and function.
10.1097/BRS.0b013e3181da38d5
Synergetic enrichment of aggrecan in nucleus pulposus cells by scAAV6-shRNA-mediated knockdown of aggrecanase-1 and aggrecanase-2.
Experimental biology and medicine (Maywood, N.J.)
Degenerative disk disease (DDD) that aggravates structural deterioration of intervertebral disks (IVDs) can be accompanied by painful inflammation and immunopathological progressions. Current surgical or pharmacological therapies cannot repair the structure and function of IVDs. Nucleus pulposus (NP) cells are crucial for the preservation or restoration of IVDs by balancing the anabolic and catabolic factors affecting the extracellular matrix. Imbalanced anabolic and catabolic factors cause increased degradation of aggrecan. Aggrecanases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)4 and ADAMTS5 are the main degrading enzymes of aggrecan. Previously, we characterized adeno-associated virus (AAV6) as the most suitable serotype with marked NP cellular tropism and demonstrated that ADAMTS4 could be silenced by self-complementary adeno-associated virus grade 6 small helix ribonucleic acid (scAAV6-shRNA) in NP cells of degeneration grade III, which resulted in enrichment of aggrecan. Nonetheless, neither scAAV6-shRNA-mediated inhibition of ADAMTS5 nor joint inhibitions of ADAMTS4 and ADAMTS5 have been investigated, although both enzymes are regulated by analogous proinflammatory cytokines and have the same cleavage sites in aggrecan. Therefore, we attempted scAAV6-shRNA-mediated inhibitions of both enzymes in NP cells of degeneration grade IV to increase efficacies in treatments of DDD. The degeneration grade of IVDs in patients was determined by magnetic resonance imaging (MRI) before surgical operations. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting ADAMTS4 or ADAMTS5. Transduced cells were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), fluorescence microscopy, flow cytometry-assisted cell sorting (FACS), MTT assay (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay), immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Joint transduction of NP cells exhibited high transduction efficacies (98.1%), high transduction units (TU) (1381 TU/Cell), and no effect on cell viability or proliferation. Above all joint treatments resulted in effective knockdown of ADAMTS4 (92.8%) and ADAMTS5 (93.4%) along with additive enrichment of aggrecan (113.9%). Treatment effects were significant for more than 56 days after transduction ( < 0.001). In conclusion, scAAV6-shRNA-mediated combined molecular therapy could be very valuable for more effective, durable, and less immunogenic treatment approaches in DDD.
10.1177/15353702231171905
Collagen type II is downregulated in the degenerative nucleus pulposus and contributes to the degeneration and apoptosis of human nucleus pulposus cells.
Lian Chengjie,Gao Bo,Wu Zizhao,Qiu Xianjian,Peng Yan,Liang Anjing,Xu Caixia,Su Peiqiang,Huang Dongsheng
Molecular medicine reports
Degenerative disc disease (DDD) is a common degenerative condition initiated mainly within the nucleus pulposus (NP). To date, the etiopathogenesis of DDD remains unclear, and because no effective therapeutic strategies are available to target its pathological processes, DDD is still treated with symptomatic interventions that are far from adequate. Collagen type II is one of the major matrix components of the NP, and is considered to be essential to NP homeostasis. However, the specific mechanisms by which collagen type II influences NP cells remain unknown. In the present study, collagen type II expression was detected using immunohistochemistry analysis and quantitative polymerase chain reaction, and it was demonstrated to be significantly downregulated in NP tissues from patients with DDD compared with nondegenerative controls. To further explore the mechanism in vitro, interleukin (IL)‑1β stimulation was used to induce degeneration of a human NP cell line. IL‑1β stimulation upregulated both the mRNA and protein levels of the catabolic markers matrix metalloproteinase 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), while it downregulated the anabolic makers aggrecan and collagen type II. However, addition of purified collagen type II prevented this IL‑1β‑induced metabolic disturbance of the NP cells. Furthermore, IL‑1β stimulation significantly promoted apoptosis in NP cells, while collagen type II treatment decreased the apoptotic rate and the protein levels of cleaved caspase‑3. In conclusion, collagen type II exhibited protective effects in suppressing NP cell degeneration through its anticatabolic, proanabolic and antiapoptotic effects, suggesting that it may be a promising therapeutic agent for the prevention and treatment of DDD.
10.3892/mmr.2017.7178
Regenerative Capability of Human Nucleus Pulposus Cells in Degenerated Disc Under Hydrostatic Pressure Mimicking Physiologically Relevant Intradiscal Pressure In Vitro.
Spine
STUDY DESIGN:Isolated human nucleus pulposus (hNP) cells from the degenerated intervertebral disc (IVD) were incubated under hydrostatic pressure (HP) and evaluated for regenerative potential. OBJECTIVES:To characterize metabolic turnover in hNP cells isolated from degenerated IVDs classified by Pfirrmann grade under physiologically relevant HP at high osmolality in vitro. SUMMARY OF BACKGROUND DATA:We demonstrated that bovine caudal nucleus pulposus cells isolated from healthy cows produced more extracellular matrix under cyclic HP followed by constant pressure (mimicking physiological intradiscal pressure in humans) than under no pressure in vitro. We assessed the effects of pressure on human degenerated cells isolated under the same regimen of pressure used for bovine cells. MATERIALS AND METHODS:hNP cells isolated from discarded tissue classified as Pfirrmann grade 2 to 3 (n = 13: age, 46.7 ± 14.0) and grade 4 (n = 13: age, 53.0 ± 11.5) were incubated under cyclic HP at 0.2 to 0.7 MPa, 0.5 Hz for 2 days followed by constant pressure at 0.3 MPa for 1 day, repeated twice over 6 days. The gene expression and immunohistology of matrix molecules and catabolic and anticatabolic proteins were evaluated. RESULTS:Aggrecan and collagen type II expression were significantly more upregulated under HP in grades 2 to 3 than in grade 4 tissues (both, P < 0.01). Linear regression analysis showed a positive correlation between matrix metalloproteinase 13 and tissue inhibitor for metalloproteinase 2 expression in grades 2 to 3, whereas a negative correlation was found in grade 4 ( P < 0.05). Immunohistological staining revealed the activation of a mechanoreceptor, transient receptor potential vanilloid 4, under HP. CONCLUSIONS:Resident cells in mild-moderate degenerated discs classified as Pfirrmann grade 2 to 3 have the potential to promote extracellular matrix production and maintain adequate cell viability under physiological spinal loading. RELEVANCE:This study explored the potential of degenerated remnant nucleus pulposus cells under a physiological environment, possibly leading to establishing strategies for IVD regeneration.
10.1097/BRS.0000000000004530
Positive feedback loop of interleukin-1beta upregulating production of inflammatory mediators in human intervertebral disc cells in vitro.
Jimbo Kotaro,Park Jin Soo,Yokosuka Kimiaki,Sato Kimiaki,Nagata Kensei
Journal of neurosurgery. Spine
OBJECT:Interleukin-1beta (IL-1beta) induces neurological symptoms in intervertebral disc herniation (IDH). Recently, the existence of a positive feedback loop of IL-1beta, which encourages an inflammatory reaction or degeneration in the cells of tendon, has been reported. The authors hypothesized that there is a positive feedback loop of IL-1beta in the cells of IDH. METHODS:Eight human intervertebral disc specimens were harvested during spinal surgery for lumbar disc herniation. The cells were stimulated in serum-free medium with or without exogenous IL-1beta. The messenger RNA (mRNA) was extracted for reverse-transcription polymerase chain reaction (PCR) and real-time PCR to quantify the mRNA of endogenous IL-1beta, IL-6, cyclooxygenase-2 (COX-2), and matrix metalloproteinases (MMPs). The cells were then stimulated in serum-free medium with or without exogenous IL-1beta, and then exogenous IL-1beta was removed. After 2, 4, and 6 days, the medium was collected, and enzyme-linked immunosorbent assay was used to measure the protein concentration of endogenous IL-1beta. The mRNA expressions of endogenous IL-1beta, IL-6, COX-2, and MMPs were increased significantly depending on the concentration of exogenous IL-1beta. The protein concentration of endogenous IL-1beta was increased over time. CONCLUSIONS:There was a positive feedback loop of IL-1beta in the cells of IDH. Furthermore, the productions of IL-6, COX-2, MMP-1, and MMP-3 were upregulated as a result of the increasing concentration of IL-1beta in a positive feedback loop of IL-1beta. The authors concluded that this positive feedback loop of IL-1beta upregulated the production of mediators and thus can cause cessation of symptoms in IDH.
10.3171/spi.2005.2.5.0589
Melatonin inhibits nucleus pulposus (NP) cell proliferation and extracellular matrix (ECM) remodeling via the melatonin membrane receptors mediated PI3K-Akt pathway.
Li Zheng,Li Xingye,Chen Chong,Chan Matthew T V,Wu William Ka Kei,Shen Jianxiong
Journal of pineal research
Pinealectomy in vertebrates accelerated intervertebral disk degeneration (IDD). However, the potential mechanisms, particularly melatonin's role, are still to be clarified. In this study, for first time, melatonin membrane receptors of MT1 and MT2 were found to be present in the human intervertebral disk tissues and nucleus pulposus (NP) cells, respectively. Melatonin treatment significantly inhibited NP cell proliferation in dose-dependent manner. Accordingly, melatonin down-regulated gene expression of cyclin D1, PCNA, matrix metallopeptidase-3, and matrix metallopeptidase-9 and upregulated gene expression of collagen type II alpha 1 chain and aggrecan in NP cells. These effects of melatonin were blocked by luzindole, a nonspecific melatonin membrane receptor antagonist. Signaling pathway analysis indicated that in the intervertebral disk tissues and NP cells, melatonin acted on MT1/2 and subsequently reduced phosphorylation of phosphoinositide 3-kinase p85 regulatory subunit, phosphoinositide-dependent kinase-1, and Akt. The results indicate that melatonin is a crucial regulator of NP cell function and plays a vital role in prevention of IDD.
10.1111/jpi.12435
[Salvianolic acid A contributes to cartilage endplate cell restoration by regulating miR-940 and miR-576-5p].
Zhongguo gu shang = China journal of orthopaedics and traumatology
OBJECTIVE:To investigate whether Salvianolic acid A (SAA) can restore cartilage endplate cell degeneration of intervertebral discs and to identify the mechanism via regulation of micro-RNA. METHODS:Cartilage endplate cells were isolated from lumbar intervertebral disc surgical samples and were treated with serum containing a series of concentrations of SAA (2, 5, and 10 ?M) for 24, 48, and 72 h to identify a proper dose and treatment time of SAA. The effect SAA on interlenkin-1β (IL-1β)-induced extracellular matrix degradation of cartilage endplate cells were analyzed by Alcian blue staining and assessment of the expression levels of ADAMTS-5, MMP3 and Col2a1. Further, the potential target miRNAs were preliminarily screened by micro-RNA sequencing combining qRT-PCR and Western blot, and then, the miRNAs mimics and inhibitors were used to verify the regulatory effect of SAA on potential target miRNAs. RESULTS:The 10 μM SAA treatment for 48 h significantly enhanced the viability of cartilage endplate cells, and increased Col2a1 expression and glycosaminoglycan accumulation that were repressed by IL-1β, and reduced the effect of IL-1β on ADAMTS-5, and MMP3. Screening analysis based on micro-RNA sequencing and Venny analysis identified the downstream micro-RNAs, including miR-940 and miR-576-5p. Then, the miR-940-mimic or miR-576-5p-mimic were transfected into CEPCs. Compared with the SAA group, the expression of ADAMTS-5 and MMP3 increased significantly and the expression of COL2A1 obviously decreased after overexpression of miR-940 or miR-576-5p in CEPCs. CONCLUSION:Salvianolic acid A attenuated the IL-1β-induced extracellular matrix degradation of cartilage endplate cells by targeting regulate the miR-940 and the miR-576-5p.
10.12200/j.issn.1003-0034.2023.10.014
Involvement of acid-sensing ion channel 1a in matrix metabolism of endplate chondrocytes under extracellular acidic conditions through NF-κB transcriptional activity.
Cell stress & chaperones
Acidic conditions are present in degenerated intervertebral discs and are believed to be responsible for matrix breakdown. Acid-sensing ion channel 1a (ASIC1a) is expressed in endplate chondrocytes, and its activation is associated with endplate chondrocyte apoptosis. However, the precise role of ASIC1a in regulating the matrix metabolic activity of endplate chondrocytes in response to extracellular acid remains poorly understood. Aggrecan (ACAN), type II collagen (Col2a1), and matrix metalloproteinase (MMP) expressions were determined using reverse transcription (RT)-PCR and Western blot. ASIC1a was knocked down by transfecting endplate chondrocytes with ASIC1a siRNA. MMP activity and NF-κB transcriptional activity were measured. NF-κB transcriptional activity was assessed by examining cytosolic phosphorylated IκBα and nuclear phosphorylated p65 levels. Extracellular acidic solution (pH 6.0) resulted in a decrease in ACAN and Co12a1 expressions and an increase in MMP-1, MMP-9, and MMP-13 expressions, as well as in MMP activity; while ASIC1a siRNA blocked these effects. In addition, acid-induced increase in cytosolic levels of phosphorylated IκBα and nuclear levels of phosphorylated p65 in endplate chondrocytes were inhibited by ASIC1a siRNA. ASIC1a is involved in matrix metabolism of endplate chondrocytes under extracellular acidic conditions via NF-κB transcriptional activity.
10.1007/s12192-015-0643-7
Interactions between the MMP-3 gene rs591058 polymorphism and occupational risk factors contribute to the increased risk for lumbar disk herniation: A case-control study.
Luo Yongjun,Wang Jiaxing,Pei Jie,Rong Yuluo,Liu Wei,Tang Pengyu,Cai Weihua,Yin GuoYong
Journal of clinical laboratory analysis
OBJECTIVE:Lumbar disk herniation (LDH) is a complex condition based on lumbar disk degeneration (LDD). Previous studies have shown that genetic factors are highly associated with the severity and risk for LDH. This case-control study was aimed to evaluate the association between the matrix metalloproteinase (MMP)-3 gene rs591058 C/T polymorphism and LDH risk in a southern Chinese population. METHODS:A total of 231 LDH patients and 312 healthy controls were recruited in this study. Genotyping was analyzed using a standard polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). RESULTS:It was observed that TT genotype or T allele carriers of the MMP-3 gene rs591058 C/T polymorphism was more likely associated with an increased risk for LDH. Subgroup analyses showed the following characteristics increased the risk for LDH: female sex; cigarette smoking; and alcohol consumption. Furthermore, individuals with high whole body vibration, bending/twisting, and lifting were associated with an increased risk for LDH. CONCLUSION:Taken together, these data indicated that the MMP-3 gene rs591058 C/T polymorphism was associated with an increased risk for LDH. The MMP-3 gene rs591058 C/T polymorphism might serve as a clinical indicator and marker for LDH risk in the Chinese population.
10.1002/jcla.23273
Anti-catabolic effect of OP-1 in chronically compressed intervertebral discs.
Chubinskaya Susan,Kawakami Mamoru,Rappoport Lev,Matsumoto Takuji,Migita Nami,Rueger David C
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Experimental animal models of disc degeneration have been used to assess the biomechanical behavior, biochemical composition, and biological changes in the intervertebral discs. The objective of our study was to evaluate the anabolic and anti-catabolic effects of intradiscal injection of Osteogenic Protein-1 (OP-1) by histology and immunohistochemistry in disc degeneration model. Thirty-four rats were divided into five groups: intact control; sham control; compressed nucleus pulposus (NP) injected with saline; and two OP-1 groups: COP-1 group (compression was continued after intradiscal OP-1 injection) and ROP-1 group (compression was released at the time of OP-1 injection). Anabolic and anti-catabolic effects of OP-1 were evaluated by histology and immunohistochemistry with the following antibodies: anti-pro- and anti-mature OP-1, anti-MMP-13, anti-aggrecanase, anti-substance P, anti-tumor necrosis factor-alpha (TNF-alpha), and anti-interleukin-1beta (IL-1beta). The OP-1 injection to the degenerative disc stimulated an anabolic response characterized by the restoration of the normal morphology of the disc, increased Safranin O staining in the NP, extention of the extracellular matrix, and stimulation of endogenous OP-1 synthesis in the NP, annulus fibrosis (AF), and end-plate. The anti-catabolic effect of OP-1 was documented by reduced immunostaining for aggrecanase, MMP-13, substance P, TNF-alpha, and IL-1beta. This study confirmed the anti-catabolic activity of OP-1 as demonstrated previously in human articular cartilage and provided critical evidence for the potential of OP-1 therapy in the treatment of disc degeneration. Because substance P is a neuropeptide linked with inflammation and pain, a reduction in the level of this protein may support our previously reported results on the effect of OP-1 on pain-related behavior.
10.1002/jor.20339
Naringin Protects Against Interleukin 1β (IL-1β)-Induced Human Nucleus Pulposus Cells Degeneration via Downregulation Nuclear Factor kappa B (NF-κB) Pathway and p53 Expression.
Gao Gang,Chang Feng,Zhang Ting,Huang Xinhu,Yu Chen,Hu Zhaolin,Ji Mingming,Duan Yufen
Medical science monitor : international medical journal of experimental and clinical research
<strong>BACKGROUND</strong> Low back pain (LBP) is regarded as a frequent disease that causes disability. We aimed to explore the effect of naringin on intervertebral disc degeneration (IDD) in IL-1ß-induced human nucleus pulposus (NP) cells and its corresponding molecular mechanisms. <strong>MATERIAL AND METHODS</strong> Human NP cells were identified by toluidine blue and Safranin O staining. Cell viability was determined by MTT assay. The expression levels of matrix metalloproteinases (MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, collagen II, aggrecan), inflammatory genes (tumor necrosis factor [TNF]-alpha, interleukin [IL]-6), kappa B kinase alpha (IkappaBalpha), p65 and p53 were determined by quantitative real-time polymerase chain reaction (qPCR) and western blotting. Immunofluorescence study was performed to detect the position and expression of p65 protein in IL-1ß-induced human NP cells. <strong>RESULTS</strong> Human NP cells were successfully separated from intervertebral disc tissue. We found that naringin could significantly reduce the expressions of matrix metalloproteinases (MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5) and inflammatory genes in IL-1ß-stimulated human NP cells, while collagen II and aggrecan were increased at mRNA and protein level. Immunofluorescence showed that naringin pretreatment decreased the p65 protein expression in the nucleus and suppressed the phosphorylation of IkappaBalpha and p65. <strong>CONCLUSIONS</strong> These results demonstrated that naringin could attenuate matrix metalloproteinase catabolism and inflammation in IL-1ß-treated human nucleus pulposus cells via downregulating NF-kappaB pathway and p53 expression, suggesting that naringin has the potential to prevent and treat IDD.
10.12659/MSM.918597
The Paracrine Effect of Degenerated Disc Cells on Healthy Human Nucleus Pulposus Cells Is Mediated by MAPK and NF-κB Pathways and Can Be Reduced by TGF-β1.
Cai Feng,Zhu Lei,Wang Feng,Shi Rui,Xie Xin-Hui,Hong Xin,Wang Xiao-Hu,Wu Xiao-Tao
DNA and cell biology
Inflammation is thought to have a major role in the pathogenesis of disc degeneration. Studies have shown that nucleus pulposus cells (NPCs) respond to one or two specific cytokines by regulating cell proliferation or matrix synthesis. However, the effects of a cocktail of factors secreted by degenerated disc cells on transplanted exogenous healthy NPCs remain unknown. Concentrations of multiple cytokines in degenerated disc tissue-conditioned medium (dCM) were measured using enzyme-linked immunosorbent assay (ELISA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and Ki67 immunofluorescence staining were used to evaluate the proliferation of cells in dCM. The function of exogenous NPCs cultured in dCM was evaluated by examining catabolic markers (ADAMTS-4, ADAMTS-5, MMP-1, MMP-3, and MMP-13), anabolic markers (TIMP-1, TIMP-2, and TIMP-3), and the extracellular matrix protein-aggrecan (ACAN) and collagen II (COL2)-expression with real time polymerase chain reaction (RT-PCR). Mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) pathway activation was observed using Western blotting. Finally, we examined the role of transforming growth factor (TGF)-β1 in reducing dCM-mediated exogenous NPC dysfunction. Levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-1α, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, interferon-γ (IFN-γ), and prostaglandin E2 (PGE2) were higher and TGF-β1 levels were lower in dCM compared with the control medium. Treatment with dCM increased the proliferation of healthy NPCs. NPCs exhibited significantly higher expression of ADAMTS-4, ADAMTS-5, MMP-1, MMP-3, and MMP-13 and decreased TIMP-2, ACAN, and COL2 expression in the dCM group in a dose- and time-dependent manner. Treatment with dCM moderately increased TIMP-1 expression and had no effect on TIMP-3 mRNA levels. The MAPK and NF-κB pathways were implicated in dCM-mediated responses of healthy NPCs. TGF-β1 partially reversed the dCM-mediated NPC dysfunction. Increased levels of inflammatory factors and decreased TGF-β1 levels in dCM suggest an inflammatory environment in degenerated disc tissue. The catabolic effect of dCM on human healthy NPCs is mediated by MAPK and NF-κB pathways and can be reduced by TGF-β1.
10.1089/dna.2016.3230
Propionibacterium acnes induces cartilaginous endplate degeneration by promoting MIF expression via the NF-κB pathway.
Zhang Ying,Wang Yuting,Yuan Yanyan,Lin Yeting,Lin Binbin,Zhou Haiyan
Journal of orthopaedic surgery and research
BACKGROUND:Propionibacterium acnes (P. acnes) is a novel pathogenic factor that contributes to cartilaginous endplate (CEP) degeneration. However, the underlying mechanism of P. acnes-induced CEP degeneration remains unclear. The objective of this study is to investigate the underlying mechanism of P. acnes-induced CEP degeneration. METHODS:We first examined MIF expression in degenerated human CEP samples by immunohistochemistry. We developed a P. acnes-induced rat model and detected MIF expression using immunohistochemistry. Additionally, we investigated the mechanism of P. acnes-induced CEP degeneration in CEP cells using western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RESULTS:We found that compared with the normal human CEP, the expression of MIF was increased in the degenerated human CEP. In a rat model, P. acnes induced CEP degeneration and upregulated MIF expression significantly. More importantly, we revealed the underlying mechanism of P. acnes-induced CEP degeneration in the rat CEP cells. Firstly, P. acnes induced the expression of MIF in a concentration-dependent manner. Then, MIF upregulated the expression of MMP-13 and promoted the secretion of IL-6 and IL-1β. Finally, P. acnes may promote MIF expression via NF-κB pathway rather than ERK1/2 pathway. CONCLUSION:P. acnes-induced MIF expression via NF-κB pathway may be the underlying mechanism of CEP degeneration.
10.1186/s13018-020-01714-6
Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue.
Séguin Cheryle A,Pilliar Robert M,Roughley Peter J,Kandel Rita A
Spine
STUDY DESIGN:This study examines changes in the production of extracellular matrix molecules as well as the induction of tissue degradation in in vitro formed nucleus pulposus (NP) tissues following incubation with tumor necrosis factor (TNF)alpha. OBJECTIVE:To characterize the response of NP cells to TNF-alpha, a proinflammatory cytokine present in herniated NP tissues. SUMMARY OF BACKGROUND DATA:TNF-alpha is a proinflammatory cytokine expressed by NP cells of degenerate intervertebral discs. It is implicated in the pain associated with disc herniation, although its role in intervertebral disc degeneration remains poorly understood. METHODS:In vitro formed NP tissues were treated with TNF-alpha (up to 50 ng/mL) over 48 hours. Tissues were assessed for histologic appearance, proteoglycan and collagen contents, as well as proteoglycan and collagen synthesis. Reverse transcriptase polymerase chain reaction was used to determine the effect of TNF-alpha on NP cell gene expression. Proteoglycan degradation was assessed by immunoblot analysis. RESULTS:At doses of 1-5 ng/mL, TNF-alpha induced multiple cellular responses, including: decreased expression of both aggrecan and type II collagen genes; decreases in the accumulation and overall synthesis of aggrecan and collagen; increased expression of MMP-1, MMP-3, MMP-13, ADAM-TS4, and ADAM-TS5; and induction of ADAM-TS dependent proteoglycan degradation. Within 48 hours, these cellular responses resulted in NP tissue with only 25% of its original proteoglycan content. CONCLUSIONS:Because low levels of TNF-alpha, comparable to those present physiologically, induced NP tissue degradation, this suggests that TNF-alpha may contribute to the degenerative changes that occur in disc disease.
Nerve growth factor increases MMP9 activity in annulus fibrosus cells by upregulating lipocalin 2 expression.
Kao Ting-Hsien,Peng Yi-Jen,Salter Donald M,Lee Herng-Sheng
European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society
PURPOSE:Nerve growth factor (NGF) expression and activity is important in chronic lower back pain but may also act as a pro-catabolic factor in the pathogenesis of intervertebral disc (IVD) degeneration. Lipocalin 2 (Lcn2) expression in IVD was upregulated by NGF stimulation in our previous study. The current study was undertaken to identify potential mechanisms of the latter effect including potential interactions between Lcn2 and matrix metalloproteinase 9 (MMP9). METHODS:Rat annulus fibrosus (AF) cells were stimulated by NGF and subjected to microarray analysis, subsequent real-time PCR, western immunoblotting, and immunofluorescence. Cells were treated with NGF in the absence or presence of the NGF inhibitor Ro 08-2750. Zymography and functional MMP9 assays were used to determine MMP9 activity, whilst the dimethyl-methylene blue assay was used to quantify the release of glycosaminoglycans (GAGs) reflecting catabolic effects following NGF treatment. Immunoprecipitation with immunoblotting was used to identify interactions between MMP9 and Lcn2. RESULTS:Increased expression of Lcn2 gene and protein following NGF stimulation was confirmed by microarray analysis, real-time PCR, western blot and immunofluorescence. Zymography showed that NGF enhanced 125-kDa gelatinase activity, identified as a Lcn2/MMP9 complex by immunoprecipitation and immunoblotting. Functional assays showed increased MMP9 activity and GAG release in the presence of NGF. The effects of NGF were neutralized by the presence of Ro 08-2750. CONCLUSIONS:NGF upregulates Lcn2 expression and increases MMP9 activity in AF cells; processes which are likely to potentiate degeneration of AF tissue in vivo. Anti-NGF treatment may have benefit for management of pain relief and slowing down progression of AF tissue degeneration.
10.1007/s00586-014-3675-2
Effects of photobiomodulation on annulus fibrosus cells derived from degenerative disc disease patients exposed to microvascular endothelial cells conditioned medium.
Hwang Min Ho,Lee Jae Won,Son Hyeong-Guk,Kim Joohan,Choi Hyuk
Scientific reports
Intervertebral disc (IVD) degeneration with chronic low back pain is associated with neo-vascularisation into the deeper IVD regions. During this process, endothelial cells (ECs), which are primarily responsible for angiogenesis, interact with the adjacent annulus fibrosus (AF) cells, which are the first line of defence against the invasion of vascular structures into deeper IVD regions. However, the accumulation of inflammatory and catabolic enzymes that results from this interaction promotes matrix degradation and an inflammatory response. Thus, regulating the production of these mediators and catabolic enzymes could ameliorate IVD degeneration. Photobiomodulation (PBM) therapy is a non-invasive stimulation known to have biologically beneficial effects on wound healing, tissue repair, and inflammation. Here, we examined the effects of PBM, administered at various wavelengths (645, 525, and 465 nm) and doses (16, 32, and 64 J/cm), on EC-stimulated human AF cells. Our results show that PBM selectively inhibited the EC-mediated production of inflammatory mediators, catabolic enzymes, and neurotrophins by human AF cells in a dose- and wavelength-dependent manner. These results suggest that PBM could be a superior and advanced treatment strategy for IVD degeneration.
10.1038/s41598-020-66689-0
Collagen-derived N-acetylated proline-glycine-proline upregulates the expression of pro-inflammatory cytokines and extracellular matrix proteases in nucleus pulposus cells via the NF-κB and MAPK signaling pathways.
Feng Chencheng,He Jinyue,Zhang Yang,Lan Minghong,Yang Minghui,Liu Huan,Huang Bo,Pan Yong,Zhou Yue
International journal of molecular medicine
N-acetylated proline-glycine-proline (N-Ac-PGP) is a chemokine involved in inflammatory diseases and is found to accumulate in degenerative discs. N-Ac-PGP has been demonstrated to have a pro-inflammatory effect on human cartilage endplate stem cells. However, the effect of N-Ac-PGP on human intervertebral disc cells, especially nucleus pulposus (NP) cells, remains unknown. The purpose of this study was to investigate the effect of N-Ac-PGP on the expression of pro-inflammatory factors and extracellular matrix (ECM) proteases in NP cells and the molecular mechanism underlying this effect. Therefore, Milliplex assays were used to detect the levels of various inflammatory cytokines in conditioned culture medium of NP cells treated with N-Ac-PGP, including interleukin-1β (IL-1β), IL-6, IL-17, tumor necrosis factor-α (TNF-α) and C-C motif ligand 2 (CCL2). RT-qPCR was also used to determine the expression of pro-inflammatory cytokines and ECM proteases in the NP cells treated with N-Ac-PGP. Moreover, the role of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in mediating the effect of N-Ac-PGP on the phenotype of NP cells was investigated using specific signaling inhibitors. Milliplex assays showed that NP cells treated with N-Ac-PGP (10 and 100 µg/ml) secreted higher levels of IL-1β, IL-6, IL-17, TNF-α and CCL2 compared with the control. RT-qPCR assays showed that NP cells treated with N-Ac-PGP (100 µg/ml) had markedly upregulated expression of matrix metalloproteinase 3 (MMP3), MMP13, a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4), ADAMTS5, IL-6, CCL-2, CCL-5 and C-X-C motif chemokine ligand 10 (CXCL10). Moreover, N-Ac-PGP was shown to activate the MAPK and NF-κB signaling pathways in NP cells. MAPK and NF-κB signaling inhibitors suppressed the upregulation of proteases and pro-inflammatory cytokines in NP cells treated with N-Ac-PGP. In conclusion, N-Ac-PGP induces the expression of pro-inflammatory cytokines and matrix catabolic enzymes in NP cells via the NF-κB and MAPK signaling pathways. N-Ac-PGP is a novel therapeutic target for intervertebral disc degeneration.
10.3892/ijmm.2017.3005
Simvastatin Inhibits IL-1β-Induced Apoptosis and Extracellular Matrix Degradation by Suppressing the NF-kB and MAPK Pathways in Nucleus Pulposus Cells.
Tu Ji,Li Wentian,Zhang Yukun,Wu Xinghuo,Song Yu,Kang Liang,Liu Wei,Wang Kun,Li Shuai,Hua Wenbin,Yang Cao
Inflammation
Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway. Some studies have shown that statins may have the potential to inhibit intervertebral disk (IVD) degeneration (IDD). Interleukin (IL)-1β, a catabolic cytokine, is a key regulator of IDD. This study aimed to investigate the mechanism underlying the effect of simvastatin on IDD. The viability of nucleus pulposus (NP) cells was determined by the methyl-thiazolyl-tetrazolium (MTT) assay. The apoptosis of NP cells was measured by flow cytometric analysis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and western blotting of relevant apoptotic proteins. The protein levels of catabolic factors and anabolic factors were determined by western blotting. The cells were stimulated with IL-1β in the absence or presence of simvastatin to investigate the effects on matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, type II collagen, and aggrecan expression. Our findings indicate that simvastatin considerably inhibited IL-1β-induced apoptosis in NP cells. We also found that simvastatin attenuated IL-1β-induced expression and MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 activities and also reduced the decrease in type II collagen and aggrecan expression. In addition, simvastatin considerably suppressed the nuclear translocation and activation of nuclear factor-kappa B (NF-KB) by inhibiting p65 phosphorylation and translocation and blocking inhibitor kB-α degradation. It also inhibited MAPK pathway activation by blocking c-Jun N-terminal kinase (JNK), p38, and ERK phosphorylation. The results of our study revealed that simvastatin is a potential agent for IDD prevention and treatment.
10.1007/s10753-017-0516-6
Dynamic Compression Effects on Immature Nucleus Pulposus: a Study Using a Novel Intelligent and Mechanically Active Bioreactor.
Li Pei,Gan Yibo,Wang Haoming,Zhang Chengmin,Wang Liyuan,Xu Yuan,Song Lei,Li Songtao,Li Sukai,Ou Yangbin,Zhou Qiang
International journal of medical sciences
BACKGROUND:Previous cell culture and animal in vivo studies indicate the obvious effects of mechanical compression on disc cell biology. However, the effects of dynamic compression magnitude, frequency and duration on the immature nucleus pulposus (NP) from an organ-cultured disc are not well understood. OBJECTIVE:To investigate the effects of a relatively wide range of compressive magnitudes, frequencies and durations on cell apoptosis and matrix composition within the immature NP using an intelligent and mechanically active bioreactor. METHODS:Discs from the immature porcine were cultured in a mechanically active bioreactor for 7 days. The discs in various compressive magnitude groups (0.1, 0.2, 0.4, 0.8 and 1.3 MPa at a frequency of 1.0 Hz for 2 hours), frequency groups (0.1, 0.5, 1.0, 3.0 and 5.0 Hz at a magnitude of 0.4 MPa for 2 hours) and duration groups (1, 2, 4 and 8 hours at a magnitude of 0.4 MPa and frequency of 1.0 Hz) experienced dynamic compression once per day. Discs cultured without compression were used as controls. Immature NP samples were analyzed using the TUNEL assay, histological staining, glycosaminoglycan (GAG) content measurement, real-time PCR and collagen II immunohistochemical staining. RESULTS:In the 1.3 MPa, 5.0 Hz and 8 hour groups, the immature NP showed a significantly increase in apoptotic cells, a catabolic gene expression profile with down-regulated matrix molecules and up-regulated matrix degradation enzymes, and decreased GAG content and collagen II deposition. In the other compressive magnitude, frequency and duration groups, the immature NP showed a healthier status regarding NP cell apoptosis, gene expression profile and matrix production. CONCLUSION:Cell apoptosis and matrix composition within the immature NP were compressive magnitude-, frequency- and duration-dependent. The relatively high compressive magnitude or frequency and long compressive duration are not helpful for maintaining the healthy status of an immature NP.
10.7150/ijms.13747
Interaction between C/EBPβ and RUNX2 promotes apoptosis of chondrocytes during human lumbar facet joint degeneration.
Journal of molecular histology
The pathophysiological changes in cartilage are a crucial feature of lumbar facet joint (LFJ) degeneration and arthritis. However, the molecular mechanism of human LFJ degeneration remains largely defined. This study aimed to examine the changes in chondrocytes at different stages of degenerative LFJ using hematoxylin and eosin and Safranin O staining. The significant loss of chondrocytes in grades 2 and 3 of LFJs was observed. The expression levels of CCAAT enhancer binding protein β (C/EBPβ), Runt-related transcription factor 2 (RUNX2), and matrix metalloproteinase 13 (MMP13) also increased with the aggravation of degeneration (4.89, 5.77, and 6.3 times by Western blot). In vitro, chondrocytes scraped from the LFJs during surgery were stimulated by interleukin (IL)-1β to establish the injury model. The association of C/EBPβ and RUNX2 with active caspase-3 on chondrocytes was analyzed. The high expression level of C/EBPβ, RUNX2, and MMP13 was consistent with that of caspase-3, which reached a peak after 36 h of stimulation. Immunofluorescence suggested that C/EBPβ, RUNX2, and MMP13 co-labeled with active caspase-3. Moreover, immunoprecipitation data prompted that C/EBPβ was able to interact with RUNX2. The knockdown of C/EBPβ significantly decreased the expression levels of MMP13 and active caspase-3 (2.48 and 2.89 times as detected by Western blot analysis) and inhibited chondrocyte apoptosis, which was further demonstrated using flow cytometry. Taken together, the findings of this study uncovered that C/EBPβ could interact with RUNX2 to induce chondrocyte apoptosis in human LFJ degeneration by regulating the expression of MMP13.
10.1007/s10735-020-09891-8
Effects of MMP-1 and MMP-3 gene polymorphisms on gene expression and protein level in lumbar disc herniation.
Eser B,Eser O,Yuksel Y,Aksit H,Karavelioglu E,Tosun M,Sekerci Z
Genetics and molecular research : GMR
The aim of this study was to identify the possible correlation between polymorphisms in matrix metalloproteinase (MMP)-1 and MMP-3 and their corresponding protein levels in disc tissues obtained from patients with lumbar disc herniation (LDH) using biochemical and immunohistochemical analyses. Blood and disc samples were obtained from 100 patients with LDH who underwent a lumbar microdiscectomy. Based on the radiological degeneration, the patients were diagnosed with grade 2, 3, or 4 LDH. MMP-1 -1607 1G/2G and MMP-3 -1171 5A/6A were analyzed by real-time polymerase chain reaction. The expressions of MMP-1 and MMP- 3 were detected by biochemical and immunohistochemical analyses. We found no association between the MMP-1 polymorphism and disc degeneration and MMP-1 expression. However, patients expressing the 6A/6A and 5A/6A alleles of MMP-3 -11715A/6A showed higher MMP-3 expression, compared to those expressing the 5A/5A genotype. Additionally, the radiological degeneration grades were correlated with the histological degeneration scoring. Protein levels and immunopositive cell rates of MMP-1 and MMP-3 were associated with disc degeneration grades. Moreover, the MMP-1 and MMP-3 expression and the histological and radiological scores were positively correlated and the MMP-3 -11715A/6A polymorphism was associated with MMP-3 expression in herniated disc tissues. This study is the first to investigate polymorphisms in MMP-1 and MMP-3, as well as their corresponding protein expressions. We also quantified an association between the radiological degeneration grades and MMP-1 and MMP- 3 expression. Further genomic studies on MMPs could focus on the utilization of MMP-1 and MMP-3 as markers for the prevention and treatment of this disease.
10.4238/gmr.15038669
Specific inhibitory protein Dkk-1 blocking Wnt/β-catenin signaling pathway improve protectives effect on the extracellular matrix.
Ye Shunan,Wang Jing,Yang Shuhua,Xu Weihua,Xie Mao,Han Kuijing,Zhang Bo,Wu Ziyan
Journal of Huazhong University of Science and Technology. Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban
The present study examined the role of Wnt/β-catenin signaling pathway in the degeneration of nucleus pulposus cells and the protective effect of DKK1 on nucleus pulposus cells. The model of nucleus pulposus cell degeneration was induced by intra-disc injection of TNF-α, and the expression of β-catenin protein was detected by Western blotting. The cultured rabbit nucleus pulposus cells were divided into 4 groups. In group A, the cells were cultured with normal medium and served as control group. In group B, the cells were cultured with TNF-α and acted as degeneration group. In group C, the cells were cultured with TNF-α and transfected with Adv-eGFP and was used as fluorescence control group. In group D, the cells were cultured with TNF-α and transfected with Adv-hDKK1-eGFP, serving as intervention group. The expression of type II collagen, proteoglycan, β-catenin, and MMP-13 in each group was detected by immunocytochemistry and RT-PCR. The result showed that TNF-α increased the expression of β-catenin and MMP-13, and significantly inhibited the synthesis of type II collagen and proteoglycan, which resulted in the degeneration of nucleus pulposus cells. This effect could be obviously reversed by DKK1. We are led to concluded that TNF-α could activate the Wnt/β-catenin signaling pathway, and increase the expression of MMP-13, thereby resulting in disc degeneration. Specifically blocking Wnt/β-catenin signaling pathway by DKK-1 could protect the normal metabolism of intervertebral disc tissue. The Wnt pathway plays an important role in the progression of the intervertebral disc degeneration.
10.1007/s11596-011-0577-y
Electron microscopic observation of established chondrocytes derived from human intervertebral disc hernia (KTN-1) and role of macrophages in spontaneous regression of degenerated tissues.
Tsuru M,Nagata K,Ueno T,Jimi A,Irie K,Yamada A,Nishida T,Sata M
The spine journal : official journal of the North American Spine Society
BACKGROUND CONTEXT:Biological and pathological cell processes during the degeneration of intervertebral discs are as yet poorly understood. PURPOSE:An electron microscope was used to observe disc hernia degeneration at the cellular level as expressed in extruded tissue from a human intervertebral disc and in cultured chondrocytes. The mechanism of spontaneous regression was analyzed in order to investigate the effects of homologous macrophages, and the results of this analysis may be developed into a clinical therapy. STUDY DESIGN/SETTING:Extruded tissue specimens excised during surgery on human intervertebral disc hernia and cultured chondrocytes isolated from the excised tissue were observed by means of electron microscopy. Extracellular matrix metalloproteinase-3 (MMP-3) and its antagonist, tissue inhibitor of metalloproteinases-1 (TIMP-1), were observed by means of immune electron microscopy. Macrophages confirmed by CD68 immunostaining were added to the chondrocyte culture and observed by means of electron microscopy. PATIENT SAMPLE:All control subjects and patients gave written consent to the study. OUTCOME MEASURES:KTN-1 was directly observed without culture, and nuclei degeneration, the development of chromatin granules, changes in the osmotic pressure of the nuclear membrane and rough-surfaced endoplasmic reticulum, and the development of fat droplets were observed. METHODS:Tissues excised during surgery were divided, a part of the tissues were fixed in various fixatives for electron microscopy and immune electron microscopy analysis, and the other part was treated with collagenase. In addition, chondrocytes were isolated and cultured. Human peripheral blood mononuclear cells were separated using the Ficoll method. After culturing the cells, macrophages were collected, added to the chondrocyte culture, and observed under an electron microscope. CD68 positivity of the macrophages was confirmed by CD68 immunostaining. RESULTS:Freshly isolated chondrocytes in the hernia's extruded region differed markedly from cultured chondrocytes. By means of immunoelectron microscopy, MMP-3 and TIMP-1 were localized at the endoplasmic reticulum of the cultured chondrocytes. Infiltration of macrophages among the chondrocytes was observed in the mixed culture. CONCLUSIONS:The tissue extruded from the intervertebral disc showed obvious signs of degeneration, such as changes in osmotic pressure. Macrophages were observed to be the mechanism of spontaneous regression.
10.1016/s1529-9430(01)00055-9
Insulin-Like Growth Factor 1 Activates PI3k/Akt Signaling to Antagonize Lumbar Disc Degeneration.
Liu Zuoqing,Zhou Kaihua,Fu Wenqin,Zhang Hailong
Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
BACKGROUND/AIMS:The pathogenesis of Lumbar disc degeneration (LDD) has been extensively studied in the past. In particular, a role of matrix metalloproteinase 3 (MMP3) in the disease initiation and progression has been recently reported. However, an involvement of Insulin-like growth factor 1 (IGF-I)-stimulated phosphatidylinositol-3 kinase (PI3k) / Akt signaling pathway-mediated control of MMP3 in LDD has not been acknowledged. METHODS:We examined the serum IGF-1 levels and activation of the receptor for IGF-1 (IGF-1R) in resected discs in patients with LDD, compared to the fractured discs from traumatized, non-LDD subjects as a control. We analyzed the effects of IGF-1 on the activation of IGF-1R, Akt and MMP3 in a human nucleus pulposus SV40 cell line (HNPSV). We transfected HNPSV cells with a constitutive nuclear FoxO1, and analyzed its effect on the activation of IGF-1R, Akt and MMP3. RESULTS:LDD patients had significantly lower levels of serum IGF-1, and LDD discs had significantly lower levels of activated IGF-1R. IGF-1 induced phosphorylation of IGF-1R, and then phosphorylation of its downstream factor Akt in the HNPSV cells, resulting in significantly inhibition of MMP3. Further, FoxO1 nuclear retention completely abolished the inhibitory effects of IGF-1 on MMP3 in HNPSV cells. CONCLUSION:Together, IGF-1/Akt/FoxO1/MMP3 regulatory machinery may control the development of LDD.
10.1159/000430347
MiR-210 facilitates ECM degradation by suppressing autophagy via silencing of ATG7 in human degenerated NP cells.
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Intervertebral disc degeneration (IDD) is thought to be the most common cause of low back pain. Dysregulation of microRNAs (miRNAs) is involved in the development of IDD. The aim of this study was to explore the influence of miR-210 on type II collagen (Col II) and aggrecan expression and possible mechanisms in human degenerated nucleus pulposus (NP) cells. Our results showed that miR-210 levels were significantly increased in degenerated NP tissues compared with healthy controls, and positively correlated with disc degeneration grade. By gain-of-function and loss-of-function studies in human degenerated NP cells, miR-210 was shown to inhibit autophagy and then upregulate MMP-3 and MMP-13 expression, leading to increased degradation of Col II and aggrecan. Autophagy-related gene 7 (ATG7) was identified as a direct target of miR-210. Knockdown of ATG7 by small interfering RNA (siRNA) abrogated the effects of miR-210 inhibitor on MMP-3, MMP-13, Col II and aggrecan expression. Taken together, these results suggest that miR-210 inhibits autophagy via silencing of ATG7, leading to increased Col II and aggrecan degradation in human degenerated NP cells.
10.1016/j.biopha.2017.06.048
Acidic pH conditions mimicking degenerative intervertebral discs impair the survival and biological behavior of human adipose-derived mesenchymal stem cells.
Li Hao,Liang Chengzhen,Tao Yiqing,Zhou Xiaopeng,Li Fangcai,Chen Gang,Chen Qi-xin
Experimental biology and medicine (Maywood, N.J.)
This study was designed to examine the survival and biological behavior of adipose-derived mesenchymal stem cells (ADMSCs) under an intervertebral disc (IVD)-like acidic environment. Human ADMSCs isolated from two age groups were cultured under four different pH levels (pH 7.4, 7.1, 6.8 and 6.5) which mimicked the standard condition and the normal, mildly degenerated and severely degenerated IVD. Cell viability was measured by fluorescein isothiocyanate-Annexin-V/propidium iodide staining, and cell proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. The expression of aggrecan, collagen-I, collagen-II, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-3 (TIMP-3), p53 and caspase-3 at the mRNA level was examined by realtime quantitative polymerase chain reaction, and the expression of aggrecan, collagen-I, collagen-II, MMP-2 and TIMP-3 at the protein level was measured by enzyme-linked immunosorbent assay. Acidic pH inhibited the viability and proliferation, and the expression of aggrecan, collagen-I and collagen-II of ADMSCs from both age groups. ADMSCs harvested from young and mature donors exhibited similar responses to the acidic pH, although cells from young donors appeared less sensitive to the low pH levels. The results demonstrated that acidic pH in IVD may be an important deleterious factor for ADMSC-based IVD regeneration. ADMSCs harvested from young donors may be more suitable to be utilized for the implantation into degenerated IVD, and the implantations may be more effective at an early stage of IVD degeneration when the pH of matrix acidity is higher than 6.8.
10.1258/ebm.2012.012009
Association of endothelin-1 expression and cartilaginous endplate degeneration in humans.
Yuan Wei,Zhao Ming-Dong,Yuan Feng-Lai,Che Wu,Duan Ping-Guo,Liu Yi,Dong Jian
PloS one
BACKGROUND:Inflammatory cytokines are involved in intervertebral disc (IVD) degeneration. Endothelin-1 (ET-1), a 21-amino-acid cytokine implicated with cartilage degradation, is secreted by vascular endothelial cells and also by many other cell types. The expression of ET-1 in human IVD cartilage endplate (CEP) and its role in disc degeneration have not been explored. METHODS AND FINDINGS:The expression of ET-1 in degenerated CEP was analyzed by immunohistochemical staining and Western blotting; ET-1 was demonstrated in cartilaginous endplate cells (CECs) by immunofluorescent staining. The ET-1 mRNA expression and protein production by CECs stimulated by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine, were determined by real-time PCR analysis and Western blotting, respectively. The matrix metalloprotease-1 (MMP-1), MMP-13 and tissue inhibitor of metalloproteases-1 (TIMP-1) levels in the supernatant of cultured CECs treated with ET-1 were determined using enzyme-linked immunosorbent assays. Nitric oxide (NO) release and nitric oxide synthase (NOS) activity were measured using a spectrophotometric assay. The apoptosis of CECs by ET-1 was measured by an Annexin V-FITC detection assay. The production of ET-1 in degenerated cartilage endplate was significantly higher than normal CEP. The results showed that ET-1 was expressed by CECs and modulated by TNF-α in a dose-dependent manner. ET-1 increased production of MMP-1 and MMP-13, decreased TIMP-1 production, and induced NO and NOS release by cultured CECs. The direct stimulation of CECs by ET-1 did not promote cell apoptosis. CONCLUSION:The study results suggest that ET-1 played a pivotal role in human CEP degeneration, and may be a new target for development of therapies for this condition.
10.1371/journal.pone.0060062
Differential regulation of matrix degrading enzymes in a TNFalpha-induced model of nucleus pulposus tissue degeneration.
Séguin Cheryle A,Bojarski Marla,Pilliar Robert M,Roughley Peter J,Kandel Rita A
Matrix biology : journal of the International Society for Matrix Biology
Intervertebral disc degeneration occurs commonly and is linked to persistent back pain and the development of disc herniation. The mechanisms responsible for tissue catabolism have not yet been fully elucidated. Previously we characterized an in vitro model of TNFalpha-induced nucleus pulposus degeneration, which demonstrates decreased expression of matrix macromolecules, increased expression of matrix degrading enzymes, and the activation of aggrecanase-mediated proteoglycan degradation [Seguin, C.A., Pilliar, R.M., Roughley, P.J., and Kandel, R.A. 2005. Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue. Spine 30: 1940-1948]. This study explores the intracellular pathways activated during TNFalpha-induced matrix degradation. We demonstrate that in nucleus pulposus cells, the p38 and JNK pathways regulate induction of MMP-1 and -3; p38, JNK, and NF-kappaB regulate the induction of MMP-13; and ERK regulates the up-regulation of MT1-MMP mRNA in response to TNFalpha. Induction of ADAMTS-4 and -5 mRNA occurred downstream of NF-kappaB activation. Depletion of tissue proteoglycans was mediated by ERK and NF-kappaB-dependent "aggrecanase" activity, suggesting MT1-MMP and ADAMTS-4 and -5 as effectors of TNFalpha-induced tissue catabolism.
10.1016/j.matbio.2006.07.002
High mobility group box-1 induces pro-inflammatory signaling in human nucleus pulposus cells via toll-like receptor 4-dependent pathway.
Shah Bhranti S,Burt Kevin G,Jacobsen Timothy,Fernandes Tiago D,Alipui Didier Olivier,Weber Kathryn T,Levine Mitchell,Chavan Sangeeta S,Yang Huan,Tracey Kevin J,Chahine Nadeen O
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Intervertebral disc (IVD) degeneration (DD) is associated with low back pain, the leading cause of disability worldwide. Damage-associated molecular patterns (DAMPs) that contribute to inflammation and trigger DD have not been well characterized. Extracellular high mobility group box-1 (HMGB1) protein has been implicated as a potent DAMP and pro-inflammatory stimulus in the immune system. In this study, we show that HMGB1 and IL-6 levels increase in patients with advanced DD in comparison to early DD. This study further tested the hypothesis that HMGB1 promotes inflammatory signaling driving DD in human nucleus pulposus (NP) cells and tissue. Immunofluorescence and western blot analysis confirmed the expression of HMGB1 and its extracellular release by NP cells under cell stress. Gene expression and protein quantification indicate that HMGB1 stimulates the expression IL-6 and MMP-1 in a dose-dependent manner. The contributions of toll-like receptor (TLR) -2, -4 and receptor for advanced glycation end products (RAGE) as receptors mediating HMGB1 signaling was examined using small molecule inhibitors. Inhibition of TLR-4 signaling, with TAK-242, completely abrogated HMGB1 induced IL-6 and MMP-1 expression, whereas inhibition of TLR-2, with O-vanillin, or RAGE, with FPS-ZM1, had mild inhibitory effects. HMGB1 stimulation activated NF-ĸB signaling while TAK-242 co-treatment abrogated it. Lastly, effects of HMGB1 on matrix deposition was evaluated in a 3D culture system of human NP cells. These results implicate HMGB1 as a potent DAMP that promotes inflammation in NP cells and degradation of NP tissues. TLR4-HMGB1 axis is a potential major pathway to alleviate disc inflammation and mitigate DD. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
10.1002/jor.24154
Moracin attenuates LPS-induced inflammation in nucleus pulposus cells via Nrf2/HO-1 and NF-κB/TGF-β pathway.
Gu Ronghe,Huang Zonggui,Liu Huijiang,Qing Qiwen,Zhuan Zou,Yang Lijing,Su Zhongyi,Huang Weiguo
Bioscience reports
The present study was designed to investigate the protective effect of moracin on primary culture of nucleus pulposus cells in intervertebral disc and explore the underlying mechanism. Moracin treatment significantly inhibited the LPS-induced inflammatory cytokine accumulation (IL-1β, IL-6 and TNF-α) in nucleus pulposus cells. And moracin also dramatically decreased MDA activity, and increased the levels of SOD and CAT induced by LPS challenge. Moreover, the expressions of Nrf-2 and HO-1 were decreased and the protein levels of p-NF-κBp65, p-IκBα, p-smad-3 and TGF-β were increased by LPS challenge, which were significantly reversed after moracin treatments. Moracin treatments also decreased the levels of matrix degradation enzymes (MMP-3, MMP-13) as indicated by RT-PCR analysis. However, Nrf-2 knockdown abolished these protective effects of moracin. Together, our results demonstrated the ability of moracin to antagonize LPS-mediated inflammation in primary culture of nucleus pulposus in intervertebral disc by partly regulating the Nrf2/HO-1 and NF-κB/TGF-β pathway in nucleus pulposus cells.
10.1042/BSR20191673
Fibronectin fragments and the cleaving enzyme ADAM-8 in the degenerative human intervertebral disc.
Ruel Nancy,Markova Dessislava Z,Adams Sherrill L,Scanzello Carla,Cs-Szabo Gabriella,Gerard David,Shi Peng,Anderson D Greg,Zack Marc,An Howard S,Chen Di,Zhang Yejia
Spine
STUDY DESIGN:The presence of fibronectin fragments (FN-fs) and the cleaving enzyme, A disintegrin and metalloproteinase domain-containing protein (ADAM)-8 were examined in human intervertebral disc (IVD) tissue in vitro. OBJECTIVE:To investigate the presence and pathophysiological concentration of FN-fs and their cleaving enzyme, ADAM-8, in the human IVD tissue. SUMMARY OF BACKGROUND DATA:The 29-kDa FN-f has been shown to result in extracellular matrix loss in rabbit IVDs. However, the concentration of this biologically active fragment in the degenerative human IVD tissue has previously not been determined. Furthermore, it is critical to identify the enzyme(s) responsible for FN cleavage in the IVD. METHODS:Human degenerative IVD tissues were removed during spinal surgery. A normal seeming young adult and an infant human cadaveric sample were obtained as controls. Soluble proteins were extracted, and analyzed by Western blotting using antibodies specific for the human FN neoepitope VRAA²⁷¹. A purified 29-kDa FN-f was used to allow estimation of the concentration of FN-fs in the tissues. ADAM-8, a FN-cleaving enzyme, was analyzed by Western blotting and immunostaining. RESULTS:All adult IVD tissues contain many FN-f species, but these species were absent from the infant disc tissue. Moderately degenerative discs contained the highest amount of FN-fs; the concentration was estimated to be in the nanomolar range per gram of tissue. ADAM-8, known to cleave FN resulting in the VRAA²⁷¹ neoepitope, was present in the human disc. ADAM-8 primarily localized in the pericellular matrix of the nucleus pulposus tissue, as determined by immunostaining. CONCLUSION:This is the first report that N-terminal FN-fs are consistently present in IVD tissues from adult subjects. The pathophysiological concentration of these fragments is estimated to be at nanomolar range per gram of IVD tissue. Furthermore, ADAM-8, known to cleave FN, is present at the pericellular matrix of disc cells.
10.1097/BRS.0000000000000397
Alterations in gene expression in response to compression of nucleus pulposus cells.
Sowa Gwendolyn A,Coelho J Paulo,Bell Kevin M,Zorn Andrew S,Vo Nam V,Smolinski Patrick,Niyonkuru Christian,Hartman Robert,Studer Rebecca K,Kang James D
The spine journal : official journal of the North American Spine Society
BACKGROUND CONTEXT:It is clear that mechanical forces are involved in initiating disc degeneration but also have the potential to exert beneficial effects. However, the signaling pathways initiated by mechanical stress and thresholds for these responses have not been elucidated. We have developed a metabolically active compression system with the advantages of having the ability to test cells in vitro as well as within their native matrix and control exposure to environmental factors. We hypothesized that nucleus pulposus cells would respond to compressive stress with different thresholds for alterations in catabolic and anabolic gene expression. PURPOSE:The purpose of the study was to establish the utility of a novel compression chamber and examine the effects of various magnitudes and durations of compression on nucleus pulposus inflammatory, catabolic, and anabolic gene expression. STUDY DESIGN:In vitro controlled examination of intervertebral disc cell responses to compression. METHODS:A chamber capable of imparting 0 to 20 MPa of hydrostatic compression onto nucleus pulposus cells was fabricated. Healthy rabbit nucleus pulposus cells were cultured in alginate beads and exposed to static compression at 0.7, 2, and 4 MPa for 4 or 24 hours. Gene expression analysis (real-time polymerase chain reaction) was performed to compare markers of inflammation (inducible nitric oxide synthase, cyclooxygenase-2), matrix catabolism (matrix metalloproteinase-3), and anticatabolic/anabolic metabolism (tissue inhibitor of metalloproteinase-1, aggrecan) in control and compressed cells. RESULTS:Compression resulted in magnitude- and duration-dependent changes in gene expression. Increasing magnitudes showed more anticatabolic gene expression changes, whereas increasing duration resulted in increases in procatabolic gene expression. CONCLUSION:These data demonstrate favorable effects of compression in relation to genes involved in matrix homeostasis and procatabolic gene expression in response to sustained loading levels, consistent with traumatic effects. These data provide an improved understanding of how compression affects cell signaling, which has the potential to be exploited to initiate repair and prevent matrix breakdown.
10.1016/j.spinee.2010.09.019
Nerve growth factor promotes expression of novel genes in intervertebral disc cells that regulate tissue degradation: Laboratory investigation.
Kao Ting-Hsien,Peng Yi-Jen,Tsou Hsi-Kai,Salter Donald M,Lee Herng-Sheng
Journal of neurosurgery. Spine
OBJECT:Increased neurotrophin activity in degenerative intervertebral discs (IVDs) is one potential cause of chronic low-back pain (LBP). The aim of the study was to assess if nerve growth factor (NGF) might alter gene expression of IVD cells and contribute to disc degeneration by enhancing expression or activity of factors that cause breakdown of IVD matrix. METHODS:Rat-tail IVD cells were stimulated by NGF and subjected to microarray analysis. Real-time polymerase chain reaction, Western blotting, and immunocytochemistry of rat and human IVD cells and tissues treated with NGF in vitro in the absence or presence of the NGF inhibitor Ro 08-2750 were used to confirm findings of the microarray studies. Phosphorylation of mitogen-activated protein kinase (MAPK) was used to identify cell signaling pathways involved in NGF stimulation in the absence or presence of Ro 08-2750. RESULTS:Microarray analysis demonstrated increased expression of chitinase 3-like 1 (Chi3l1), lipocalin 2 (Lcn2), and matrix metalloproteinase-3 (Mmp3) following NGF stimulation of rat IVD cells in vitro. Increased gene expression was confirmed by real-time polymerase chain reaction with a relative increase in the Mmp/Timp ratio. Increased expression of Chi3l1, Lcn2, and Mmp3 following NGF stimulation was also demonstrated in rat cells and human tissue in vitro. Effects of NGF on protein expression were blocked by an NGF inhibitor and appear to function through the extracellular-regulation kinase 1/2 (ERK1/2) MAPK pathway. CONCLUSIONS:Nerve growth factor has potential effects on matrix turnover activity and influences the catabolic/anabolic balance of IVD cells in an adverse way that may potentiate IVD degeneration. Anti-NGF treatment might be beneficial to ameliorate progressive tissue breakdown in IVD degeneration and may lead to pain relief.
10.3171/2014.6.SPINE13756
Follistatin-like protein 1 promotes inflammatory reactions in nucleus pulposus cells by interacting with the MAPK and NFκB signaling pathways.
Liu Yi,Wei Jianlu,Zhao Yunpeng,Zhang Yuanqiang,Han Yingguang,Chen Bin,Cheng Kaiyuan,Jia Jialin,Nie Lin,Cheng Lei
Oncotarget
OBJECTIVE:Follistatin-like protein 1 (FSTL1) is a well-known mediator of inflammation. Intervertebral disc disease is an inflammatory disorder. Here, we investigated the role of FSTL1 in the intervertebral discs inflammation. METHODS:Expression of FSTL1 in nucleus pulposus tissues from rats and human was determined by immunohistochemistry staining and western blot analysis. The expression levels of tumor necrosis factor-α (TNF-α), interleukin1-β (IL-1β) and matrix metalloproteinase 13 (MMP-13) in human and rat nucleus pulposus tissues were measured by immunohistochemistry staining. The mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NFκB) signaling pathways were detected by western blotting. RESULTS:FSTL1 serum levels were significantly increased in lumbar disc herniation patients and had a positive correlation with Visual Analogue Scores. Additionally, FSTL1 expression was significantly increased in extrusion group compared with protrusion and control groups. Furthermore, FSTL1 expression was significantly increased in intervertebral disc degeneration models of rats. Immunohistochemistry staining demonstrated that the levels of TNF-α, IL-1β and MMP-13 were increased in the pathogenesis of intervertebral disc disease. Recombinant human FSTL1 significantly increased the production of proinflammatory cytokines in vitro. In addition, FSTL1 promoted inflammation by activating c-Jun N-terminal kinase (JNK), extracellular regulated protein kinases 1/2(ERK1/2) and NFκB signaling. CONCLUSIONS:These data imply that FSTL1 expression was increased in the pathogenesis of intervertebral disc disease. Importantly, FSTL1 promoted inflammatory catabolism in the nucleus pulposus by activating JNK, ERK 1/2/MAPK and NFκB signaling.
10.18632/oncotarget.17400
MicroRNA-494 promotes apoptosis and extracellular matrix degradation in degenerative human nucleus pulposus cells.
Kang Liang,Yang Cao,Song Yu,Zhao Kangcheng,Liu Wei,Hua Wenbin,Wang Kun,Tu Ji,Li Shuai,Yin Huipeng,Zhang Yukun
Oncotarget
PURPOSE:This study investigated the expression and function of the microRNA-494 in intervertebral disc degeneration (IDD). RESULTS:MicroRNA-494 expression was upregulated during IDD progression; its overexpression increased the expression of ECM catabolic factors such as matrix metalloproteinase and A disintegrin and metalloproteinase with thrombospondin motif in NP cells while decreasing that of anabolic genes such as type II collagen and aggrecan; it also induced the apoptosis of NP cells, as determined by flow cytometry. These effects were reversed by microRNA-494 inhibitor treatment. SOX9 was identified as a target of negative regulation by microRNA-494. Promoter hypomethylation and NF-κB activation were associated with microRNA-494 upregulation in IDD. MATERIALS AND METHODS:MicroRNA-494 expression in degenerative nucleus pulposus (NP) tissue was assessed by quantitative real-time PCR. The effect of microRNA-494 on extracellular matrix (ECM) metabolism and NP cell apoptosis was evaluated by transfection of microRNA-494 mimic or inhibitor. The regulation of SRY-related high mobility group box (SOX)9 expression by microRNA-494 was assessed with the luciferase reporter assay, and the methylation status of the microRNA-494 promoter was evaluated by methylation-specific PCR and bisulfite sequencing PCR. The role of activated nuclear factor (NF)-κB in the regulation of microRNA-494 expression was evaluated using specific inhibitors. CONCLUSIONS:MicroRNA-494 promotes ECM degradation and apoptosis of degenerative human NP cells by directly targeting SOX9.
10.18632/oncotarget.15838
Rutin maintains redox balance to relieve oxidative stress induced by TBHP in nucleus pulposus cells.
Zhou Jian,Liu Qi,Yang Zhou,Xie Chuhai,Ling Long,Hu Hailan,Cao Yanming,Huang Yan,Hua Yue
In vitro cellular & developmental biology. Animal
Rutin is well known for its anti-inflammatory and antioxidant properties against oxidative stress. However, its protective function in nucleus pulposus cells (NPCs) remains unclear. This study was aimed to explore the effects of rutin on oxidative stress in NPCs. Primary NPCs were obtained from 1-mo-old SD rats. The NPCs were treated with tert-butyl hydrogen peroxide (TBHP) to obtain the oxidative stress, and different concentrations of rutin were used to observe its influence on the oxidative stress in NPCs. Fluorescent probe DCFH-DA was used to detect reactive oxide species (ROS). The antioxidant proteins and genes of heat shock protein 70 (HSP70), manganese superoxide dismutase (Mn-SOD), catalase, aggrecan and collagen II in NPCs were measured by western blot and real-time PCR. With the stimulation of TBHP, the content of ROS in NPCs increased significantly and showed solubility correlation. Rutin effectively reduced the accumulation of ROS in a dose-dependent manner. The antioxidant proteins of HSP70, Mn-SOD, and catalase and the matrix proteins of aggrecan and collagen II decreased remarkably with the stimulation of TBHP, while the matrix metalloproteinase-13 (MMP-13) significantly increased after TBHP intervention. Rutin boosted the expressions of the HSP70, Mn-SOD, and catalase, elevated the contents of aggrecan and collagen II, and inhibited the expression of MMP-13 in NPCs. The findings of this study suggested that rutin is able to reverse oxidative stress and maintain cellular function of NPCs, and it was indicated that rutin could be a possible therapeutic option for intervertebral disc degeneration.
10.1007/s11626-021-00581-7
Andrographolide mitigates IL‑1β‑induced human nucleus pulposus cells degeneration through the TLR4/MyD88/NF‑κB signaling pathway.
Zhang Lilian,Chen Qi,Wang Haoli,Yang Jian,Sheng Sunren
Molecular medicine reports
Intervertebral disc degeneration (IDD) is a multifactorial disease with few efficacious clinical drugs, which has been demonstrated to be associated with nucleus pulposus (NP) cells apoptosis and degeneration of the extracellular matrix (ECM). Interleukin (IL)‑1β, a common proinflammatory cytokine, is considered to be one of key regulators in IDD development. Andrographolide (AG), extracted from Andrographis paniculata, has been suggested to possess marked anti‑inflammatory properties. However, the effects of AG on IDD has not been well explored. The present study aimed to investigate the effects and the mechanisms of AG on IDD in human NP cells. NP cells were treated with IL‑1β in the absence or presence of AG to investigate the effects on cell viability, cellular apoptosis, production of ECM and matrix metalloproteinase (MMP)‑3, MMP‑9 and MMP‑13, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)‑4 and ADAMTS‑5. It was identified that IL‑1β‑induced NP cellular apoptosis was significantly inhibited by AG treatment. Furthermore, AG mitigated the IL‑1β‑induced degeneration of the ECM, which was paralleled by a decrease in MMPs and ADAMTS levels. In addition, AG exhibited marked inhibitory properties against the activation of Toll‑like receptors (TLRs), Myeloid differentiation factor 88 (MyD88) and the nuclear translocation of Nuclear factor kappa‑light‑chain‑enhancer of activated B cells (NF‑κB). Taken together, these results demonstrated that AG treatment mitigated IL‑1β‑induced NP cells degeneration through the TLR4/MyD88/NF‑κB signaling pathway, and suggested that AG may be a potential agent for IDD prevention and therapy.
10.3892/mmr.2018.9599
Investigation of the Effect of Secreted Factors from Mesenchymal Stem Cells on Disc Cells from Degenerated Discs.
Hingert Daphne,Nawilaijaroen Phonphan,Aldridge Jonathan,Baranto Adad,Brisby Helena
Cells, tissues, organs
Low back pain is experienced by a large number of people in western countries and may be caused and influenced by many different pathologies and psychosocial factors including disc degeneration. Disc degeneration involves the increased expression of proinflammatory cytokines and matrix metalloproteinases (MMPs) in the disc environment, which leads to the loss of extracellular matrix (ECM) and the viability of the native disc cells (DCs). Treatment approaches using growth factors and cell therapy have been proposed due to the compelling results that growth factors and mesenchymal stem cells (MSCs) can influence the degenerated discs. The aim of this study was to investigate the effects of conditioned media (CM) from human MSCs (hMSCs) and connective tissue growth factor (CTGF) and TGF-β on disc cells, and hMSCs isolated from patients with degenerative discs and severe low back pain. The aim was also to examine the constituents of CM in order to study the peptides that could bring about intervertebral disc (IVD) regeneration. DCs and hMSC pellets (approx.. 200,000 cells) were cultured and stimulated with hMSC-derived CM or CTGF and TGF-β over 28 days. The effects of CM and CTGF on DCs and hMSCs were assessed via cell viability, proteoglycan production, the expression of ECM proteins, and chondrogenesis in 3D pellet culture. To identify the constituents of CM, CM was analyzed with tandem mass spectrometry. The findings indicate that CM enhanced the cellular viability and ECM production of DCs while CTGF and the control exhibited nonsignificant differences. The same was observed in the hMSC group. Mass spectrometry analysis of CM identified >700 peptides, 129 of which showed a relative abundance of ≥2 (CTGF among them). The results suggest that CM holds potential to counter the progression of disc degeneration, likely resulting from the combination of all the substances released by the hMSCs. The soluble factors released belong to different peptide families. The precise mechanism underlying the regenerative effect needs to be investigated further, prior to incorporating peptides in the development of new treatment strategies for low back pain that is potentially caused by IVD degeneration.
10.1159/000506350
Fullerol nanoparticles suppress inflammatory response and adipogenesis of vertebral bone marrow stromal cells--a potential novel treatment for intervertebral disc degeneration.
Liu Qihai,Jin Li,Shen Francis H,Balian Gary,Li Xudong Joshua
The spine journal : official journal of the North American Spine Society
BACKGROUND CONTEXT:Intervertebral disc degeneration, leading to chronic back pain, is a major health problem in western societies. Vertebral bone marrow has been considered to play an important role in nutrition supply and metabolic exchange for discs. Vertebral bone marrow lesions, including fatty marrow replacement and inflammatory edema, noted on magnetic resonance imaging were first described in 1988. PURPOSE:To investigate the potential of a free radical scavenger, fullerol nanoparticles, to prevent vertebral bone marrow lesion and prevent disc degeneration by inhibiting inflammation and adipogenic differentiation of vertebral bone marrow stromal cells (vBMSCs). STUDY DESIGN/SETTING:Fullerol nanoparticle solutions were prepared to test their in vitro suppression effects on mouse vBMSC inflammation and adipogenic differentiation compared with non-fullerol-treated groups. METHODS:With or without fullerol treatment, vBMSCs from Swiss Webster mice were incubated with 10 ng/mL interleukin-1 β (IL-1 β). The intracellular reactive oxygen species (ROS) were measured with fluorescence staining and flow cytometry. In addition, vBMSCs were cultured with adipogenic medium (AM) with or without fullerol. Gene and protein expressions were evaluated by real-time polymerase chain reaction and histologic methods. RESULTS:Fluorescence staining and flow cytometry results showed that IL-1 β markedly increased intracellular ROS level, which could be prevented by fullerol administration. Fullerol also decreased the basal ROS level to 77%. Cellular production of matrix metalloproteinase (MMP)-1, 3, and 13 and tumor necrosis factor alpha (TNF-α) induced by IL-1 β was suppressed by fullerol treatment. Furthermore, adipogenic differentiation of the vBMSCs was retarded markedly by fullerol as revealed by less lipid droplets in the fullerol treatment group compared with the adipogenic group. The expression of adipogenic genes PPARγ and aP2 was highly elevated with AM but decreased on fullerol administration. CONCLUSIONS:These results suggest that fullerol prevents the catabolic activity of vBMSCs under inflammatory stimulus by decreasing the level of ROS, MMPs, and TNF-α. Also, fat formation in vBMSCs is prevented by fullerol nanoparticles, and, therefore, fullerol may warrant further in vivo investigation as an effective biological therapy for disc degeneration.
10.1016/j.spinee.2013.04.004
Inhibition of the extracellular signal-regulated kinase pathway reduces the inflammatory component in nucleus pulposus cells.
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Intervertebral disc (IVD) degeneration is a spinal disorder that triggers an inflammatory response and subsequent development of spinal pseudoarthrosis. The aim of the present study is to elucidate the role of the extracellular signal-regulated kinase (ERK) pathway in inflammation-induced IVD cells. Inflammatory human nucleus pulposus (NP) cells (NPCs) were induced using tumor necrosis factor-α and the ERK pathway was blocked using a selective molecule-based inhibitor U0126. Gene expression of catabolic and anabolic markers, proinflammatory, and NPCs markers was investigated. The enzymatic activity of matrix metalloproteinases (MMP)2/MMP9 was determined by gelatin zymography and nitrite production was assessed by Griess reaction. The NPC metabolic activity and viability were assessed using resazurin sodium-salt and live/dead assays, and subsequently, the specificity of U0126 on ERK1/2 signaling was determined. The catabolic enzyme MMP3 (p = 0.0001) and proinflammatory cytokine interleukin 6 (p = 0.036) were downregulated by U0126 in NPCs under inflammatory conditions. A significant increase of the cytokeratin 19 (p = 0.0031) was observed, suggesting a partial and possible recovery of the NP phenotype. U0126 does not seem to have an effect on prostaglandin production, aggrecanases, or other anabolic genes. We confirmed that U0126 selectively blocks the ERK phosphorylation and only affects the cell metabolic activity without the reduction of viable cells. Inhibition of ERK signaling downregulates important metalloproteinases and proinflammatory cytokines, and upregulates some NP markers, suggesting its potential to treat IVD degeneration.
10.1002/jor.25273
Autophagy-activated nucleus pulposus cells deliver exosomal miR-27a to prevent extracellular matrix degradation by targeting MMP-13.
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Although autophagy may be beneficial for maintaining the metabolic balance of the extracellular matrix (ECM) in the nucleus pulposus (NP) and its vitality under inflammation, the underlying mechanism still remains unclear. A previous study found that autophagy activation stimulated the release of exosomes in normal chondrocytes, which are located in a similar avascular environment and share many common features with those of nucleus pulposus cells (NPCs). This study explored the protective effect on matrix degradation in the NP by exosomes derived from autophagy-activated NPCs and exosomal microRNAs. NPCs-derived exosomes (NPCs-Exos) were isolated from culture medium of either normal NPCs or rapamycin-treated NPCs and quantified by nanoparticle tracking analysis. The effect of rapamycin-treated NPC-derived exosomes on NPCs were assessed by coculture with interleukin 1β (IL-1β)-stimulated NPCs. After examination of six major proteinases of the ECM, matrix metalloproteinase 13 (MMP-13) was chosen for further study. miR-27a, which targets MMP-13, was investigated through previous studies and bioinformatics tool. The levels of miR-27a were upregulated in both rapamycin-treated NPCs and their exosomes, compared to the control. When exosomal miR-27a was transferred into NPCs, it alleviated IL-1β-induced degradation of the NPC ECM by targeting MMP-13. Autophagy activation may promote the release of NPCs-derived exosomes and thereby prevent the NPC matrix from degradation. Autophagy activation also alleviates intervertebral disc degeneration (IDD), at least partly via exosomal miR-27a, which restrains MMP-13 expression under IL-1β stimulation. Our work elucidates a new mechanism for how autophagy may participate in preventing IDD, which may be a promising therapeutic strategy.
10.1002/jor.24880
Matrix modification for enhancing the transport properties of the human cartilage endplate to improve disc nutrition.
PloS one
Poor solute transport through the cartilage endplate (CEP) impairs disc nutrition and could be a key factor that limits the success of intradiscal biologic therapies. Here we demonstrate that treating the CEP with matrix metalloproteinase-8 (MMP-8) reduces the matrix constituents that impede solute uptake and thereby improves nutrient diffusion. Human CEP tissues harvested from four fresh cadaveric lumbar spines (age range: 38-66 years old) were treated with MMP-8. Treatment caused a dose-dependent reduction in sGAG, localized reductions to the amount of collagen, and alterations to collagen structure. These matrix modifications corresponded with 16-24% increases in the uptake of a small solute (376 Da). Interestingly, the effects of MMP-8 treatment depended on the extent of non-enzymatic glycation: treated CEPs with high concentrations of advanced glycation end products (AGEs) exhibited the lowest uptake compared to treated CEPs with low concentrations of AGEs. Moreover, AGE concentrations were donor-specific, and the donor tissues with the highest AGE concentrations appeared to have lower uptake than would be expected based on the initial amounts of collagen and sGAG. Finally, increasing solute uptake in the CEP improved cell viability inside diffusion chambers, which supports the nutritional relevance of enhancing the transport properties of the CEP. Taken together, our results provide new insights and in vitro proof-of-concept for a treatment approach that could improve disc nutrition for biologic therapy: specifically, matrix reduction by MMP-8 can enhance solute uptake and nutrient diffusion through the CEP, and AGE concentration appears to be an important, patient-specific factor that influences the efficacy of this approach.
10.1371/journal.pone.0215218
Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2.
Kang J D,Georgescu H I,McIntyre-Larkin L,Stefanovic-Racic M,Donaldson W F,Evans C H
Spine
STUDY DESIGN:Herniated lumbar disc specimens were obtained from patients undergoing surgical discectomy for persistent radiculopathy and cultured in vitro to determine whether various biochemical agents were being produced. OBJECTIVES:Our hypothesis is that biochemical mediators of inflammation and tissue degradation play a role in intervertebral disc degeneration and in the pathophysiology of radiculopathy. SUMMARY OF BACKGROUND DATA:Low back pain with or without radiculopathy is a significant clinical problem, but the etiology of low back pain and the exact pathophysiology of radiculopathy remain elusive. The biochemical events that occur with intervertebral disc degeneration and, in particular, the role of biochemical mediators of inflammation and tissue degradation have received sparse attention in the literature. There is some preliminary evidence that inflammatory mediators may have an important role in the pathophysiology of radiculopathy. METHODS:Eighteen herniated lumbar discs were obtained from 15 patients undergoing disc surgery. The specimens were cultured and incubated for 72 hours, and the media were collected subsequently for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, prostaglandin E2, and a variety of cytokines were performed. As a control group, eight lumbar disc specimens were obtained from four patients undergoing anterior surgery for scoliosis and traumatic burst fractures, and similar biochemical analyses were performed. RESULTS:The culture media from the herniated lumbar discs showed increased levels of matrix metalloproteinase activity compared with the control discs. Similarly, the levels of nitric oxide, prostaglandin E2, and interleukin-6 were significantly higher in the herniated discs compared with the control discs. Interleukin 1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, interleukin-1 receptor antagonist protein, and substance P were not detected in the culture media of either the herniated or control discs. CONCLUSIONS:Herniated lumbar discs were making spontaneously increased amounts of matrix metalloproteinases, nitric oxide, prostaglandin E2, and interleukin-6. These products may be involved intimately in the biochemistry of disc degeneration and the pathophysiology of radiculopathy. Their exact roles certainly need further investigation, but their mere presence implicates biochemical processes in intervertebral disc degeneration.
Downregulation of microRNA-193a-3p is involved in invertebral disc degeneration by targeting MMP14.
Journal of molecular medicine (Berlin, Germany)
UNLABELLED:Accumulating evidence suggests that microRNAs (miRNAs) play an important role in intervertebral disc degeneration (IDD), but the precise role of specific miRNAs involved in this disease remains elusive. The purpose of this study was to identify IDD-specific miRNAs, followed by functional validation of results. MiRNA expression profile was determined in nucleus pulposus (NP) tissues from patients with IDD and controls, employing Solexa sequencing and quantitative real-time PCR (qRT-PCR). Biological functions of differential expression miRNAs were further investigated in vitro and in vivo. Luciferase reporter assays and Western blotting were performed to determine miRNA targets. We identified 28 miRNAs that were differentially expressed in patients compared with controls. Following qRT-PCR confirmation, miR-193a-3p was significantly down-regulated in degenerative NP tissues. Moreover, its level was correlated with grade of disc degeneration. Through gain- and loss-of-function studies, miR-193a-3p was demonstrated to significantly promote type II collagen expression in NP cells. Knockdown of MMP14 induced effects on NP cells similar to those induced by miR-193a-3p. Bioinformatics target prediction identified MMP14 as a putative target of miR-193a-3p. Furthermore, luciferase reporter assays and Western blotting demonstrated that miR-193a-3p directly targets MMP14. MiR-193a-3p inhibited IDD in vitro and in vivo. The downregulation of miR-193a-3p induces the expression of MMP14, which promotes loss of type II collagen and thereby contributes to the development of human IDD. Our findings extend the role of miR-193a-3p in the pathogenesis of IDD and provide a potential novel therapeutic target for degenerative disc disease. KEY MESSAGES:Intervertebral disc degeneration (ICC)-specific miRNA profile generated by next generation sequencing. Downregulation of miR-193a-3p promoted loss of type II collagen by directly targeting MMP14 in IDD. miR-193a-3p inhibited IDD in vitro and in vivo. miR-193a-3p may be a promising candidate for prevention of degenerative disc disease.
10.1007/s00109-015-1371-2
Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration.
Millward-Sadler S Jane,Costello Patrick W,Freemont Anthony J,Hoyland Judith A
Arthritis research & therapy
INTRODUCTION:The aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) on protease and catabolic cytokine and receptor gene expression in normal and degenerate human nucleus pulposus cells in alginate culture. METHODS:Cells isolated from normal and degenerate nucleus pulposus regions of human intervertebral discs were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-alpha or IL-1beta for 48 hours prior to RNA extraction. Quantitative real-time polymerase chain reaction was used to assess the effect of TNF-alpha or IL-beta stimulation on the expression of matrix metalloproteinase (MMP)-3, -9 and -13, TNF-alpha, TNF receptor 1 (TNF-R1), TNF receptor 2 (TNF-R2), IL-1alpha, IL-1beta, IL-1 receptor 1 (IL-1R1) and IL-1 receptor antagonist (IL-1Ra). RESULTS:MMP-3 and MMP-9 gene expressions were upregulated to a greater level by IL-1beta than TNF-alpha. MMP-13 was upregulated by each cytokine to a similar extent. TNF-alpha and TNF-R2 expressions were upregulated by both TNF-alpha and IL-beta, whereas TNF-R1 expression was not significantly affected by either cytokine. IL-1beta and IL-1Ra expressions were significantly upregulated by TNF-alpha, whereas IL-1alpha and IL-1R1 were unchanged. CONCLUSIONS:TNF-alpha does not induce MMP expression to the same degree as stimulation by IL-1beta, but it does act to upregulate IL-1beta expression as well as TNF-alpha and TNF-R2. The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix. These results support the hypothesis that, while TNF-alpha may be an important initiating factor in matrix degeneration, IL-1beta plays a greater role in established pathological degradation.
10.1186/ar2693
The relation of matrix metalloproteinase 1, 2, 3 expressions with clinical and radiological findings in primary and recurrent lumbar disc herniations.
Ozkanli Seyma,Kaner Tuncay,Efendioglu Mustafa,Basaran Recep,Senol Mehmet,Zemheri Ebru,Gezen Ahmet Ferruh
Turkish neurosurgery
AIM:In this study, our aim was to examine if matrix metalloproteinase expressions (MMP-1, MMP-2, MMP-3) in patients operated with a lumbar disc hernia diagnosis are different in terms of clinical and neuroradiological findings. MATERIAL AND METHODS:The study included 80 patients treated with micro discectomy for lumbar disc hernia. Degeneration was scored via magnetic resonance (MR) images. MMP-1, MMP-2, and MMP-3 antibodies were used for immunohistochemical evaluation of degenerated disc materials. MMP expressions were compared between primary and recurrent cases, and correlation analysis was conducted. RESULTS:Discectomy material showed higher expression of MMP-1 and MMP-3 in cases of recurrent lumbar disc herniation than in primary herniation. There was no significant relationship between MMP expression and MR degeneration score. CONCLUSION:MMP-1 and MMP-3 expressions were significantly higher in recurrent cases in terms of magnetic resonance degeneration score. We assume that the higher co-expression of MMP-1 and MMP-3 might be used in targeted treatment regiemens in patients with recurrent LDH.
10.5137/1019-5149.JTN.11276-14.1
Human MMP28 expression is unresponsive to inflammatory stimuli and does not correlate to the grade of intervertebral disc degeneration.
Klawitter Marina,Quero Lilian,Bertolo Alessando,Mehr Marco,Stoyanov Jivko,Nerlich Andreas G,Klasen Juergen,Aebli Nikolaus,Boos Norbert,Wuertz Karin
Journal of negative results in biomedicine
BACKGROUND:MMP28 (epilysin) is a recently discovered member of the MMP (matrix metalloproteinase) family that is, amongst others, expressed in osteoarthritic cartilage and intervertebral disc (IVD) tissue. In this study the hypothesis that increased expression of MMP28 correlates with higher grades of degeneration and is stimulated by the presence of proinflammatory molecules was tested. Gene expression levels of MMP28 were investigated in traumatic and degenerative human IVD tissue and correlated to the type of disease and the degree of degeneration (Thompson grade). Quantification of MMP28 gene expression in human IVD tissue or in isolated cells after stimulation with the inflammatory mediators lipopolysaccharide (LPS), interleukin (IL)-1β, tumor necrosis factor (TNF)-α or the histondeacetylase inhibitor trichostatin A was performed by real-time RT PCR. RESULTS:While MMP28 expression was increased in individual cases with trauma or disc degeneration, there was no significant correlation between the grade of disease and MMP28 expression. Stimulation with LPS, IL-1β, TNF-α or trichostatin A did not alter MMP28 gene expression at any investigated time point or any concentration. CONCLUSIONS:Our results demonstrate that gene expression of MMP28 in the IVD is not regulated by inflammatory mechanisms, is donor-dependent and cannot be positively or negatively linked to the grade of degeneration and only weakly to the occurrence of trauma. New hypotheses and future studies are needed to find the role of MMP28 in the intervertebral disc.
10.1186/1477-5751-10-9
TSG-6 inhibits IL-1β-induced inflammatory responses and extracellular matrix degradation in nucleus pulposus cells by activating the PI3K/Akt signaling pathway.
Journal of orthopaedic surgery and research
PURPOSE:Tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6), a secreted protein associated with inflammation, is believed to possess momentous and multiple anti-inflammatory and tissue-protective properties. However, the role and potential mechanism of TSG-6 in cervical disk degeneration (CDD) are still not clear. Hence, we aimed to explore the effect of TSG-6 on CDD. METHODS:Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) or enzyme-linked immunosorbent assay was applied to detect the expression level of TSG-6 and IL-1β in normal and degenerated nucleus pulposus (NP) tissues. Then, qRT-PCR and western blot were adopted to test the TSG-6 protein expression after IL-1β treatment (10 ng/mL) in human NP cells (HNPCs). After over-expressing TSG-6, qRT-PCR was also utilized to evaluate the expression of TNF-α, IL-8, and IL-6 and the synthesis of sulfated glycosaminoglycans (sGAGs), western blot to check the expression of extracellular matrix (ECM) proteins [collagen II, aggrecan, and matrix metalloproteinase-3 (MMP-3)], pain-related molecules (CGRP, calcitonin gene-related peptide; NGF, nerve growth factor; SP, substance P), and PI3K/Akt signaling pathway-related proteins. RESULTS:Briefly speaking, TSG-6 and IL-1β expression levels were significantly increased in CDD patient tissues; and IL-1β treatment could significantly increase TSG-6 expression in HNPCs. Further research revealed that, in addition to greatly promoting sGAGs synthesis, TSG-6 over-expression also inhibited TNF-α, IL-8, and IL-6 expression and ECM degradation in IL-1β-induced HNPCs. (The collagen II and aggrecan expression was up-regulated and MMP-3 expression was down-regulated.) Furthermore, over-expression of TSG-6 could decrease the levels of CGRP, NGF, and SP protein expression and activate the PI3K/Akt signaling pathway in IL-1β-treated HNPCs. CONCLUSION:TSG-6 inhibits inflammatory responses, ECM degradation, and expression of pain-related molecules in IL-1β-induced HNPCs by activating the PI3K/Akt signaling pathway.
10.1186/s13018-022-03468-9
Impact of changes in extracellular matrix in the lumbar degenerative disc.
David G,Ciurea A V,Mitrica M,Mohan A
Journal of medicine and life
UNLABELLED:The complexity of the clinical, biochemical, hystochemical and immunologic aspects of the intervertebral disk, along with its molecular biology, justifies the object of our study on the extracellular matrix modifications in lumbar disk hernias and their impact on patient quality of life. MATERIAL AND METHOD:The research lot was composed of 50 patients, aged between 18 and 73, who have undergone lumbar disk hernia surgery. MMP-9 (metalloproteinase-9) and TIMP-1 (tissue inhibitor of matrix metalloprotease 1) have been dosed in order to study the modifications on extracellular disk matrix, and quality of life assessment was carried out both in pre-operatory and post-operatory periods. CONCLUSIONS:Patients may prevent the appearance of degenerative processes of the intervertebral disk with care and responsibility by controlling their weight, avoiding intense physical activities and ceasing to smoke.
[Expression of NF-κB in a degenerative human intervertebral disc model].
Wang X F,Zhang A P,Sun Z Y,Liu C,Kuang L H,Tian J W
Zhonghua yi xue za zhi
To investigate the changes in the expression of NF-κB signaling pathway in human degenerative intervertebral discs. From October 2014 to March 2016, 55 nucleus pulposus of surgical patients with degenerative human intervertebral disc were collected for study in Department of Orthopedic Surgey, Hospital of Zaozhuang Mining Corporation, and Department of Orthopedics, Shanghai General Hospital Affiliated Shanghai Jiaotong University, School of Medicine.The collected nucleus pulposus tissues were divided into two groups: experimental group(30) and control group(25). Cell culture observed normal and degenerative nucleus pulposus cells morphological changes; immunofluorescence observed NF-κB p65 changes in the nucleus of nucleus pulposus cells.Real-time PCR was observed changes in aggregated proteoglycans and matrix metalloproteinase gene mRNA.Finally, the use of blockers of nucleus pulposus cells were treated 24 hours, Western blot analysis the changing of p65, ADAMTS-4, MMP-13, aggregate proteoglycans and collagen Ⅱ protein expression. Compared with the experimental group, the nucleus pulposus cells in the control group had larger cell volume, abundant cytoplasm and faster growth rate.Cell immunofluorescence show Nondegenerative nucleus pulposus cells p65 protein was mainly localized in the cytoplasm, degeneration of nucleus pulposus cells p65 protein was mainly concentrated in the nucleus.RT-PCR showed degenerative group of matrix metalloproteinases (MMP-1, MMP-3, MMP-13), aggrecanase(ADAMTS-4, ADAMTS-5) and IL-6 mRNA expression was significantly higher than Nondegenerative group; aggrecan and type Ⅱ collagen expression than those without degeneration group was significantly lower.Expression of nucleus pulposus degeneration in nuclear protein p65 with the degenerative level increased gradually increased.BAY11-7082 blocked the activity of NF-κB signaling pathway, which could significantly down-regulate the expression of ADAMTS-4 and MMP-13 protein and significantly up-regulate the expression of Agg and COLⅡ protein.With the increase of BAY11-7082 concentration, gradually strengthened. The activation of the NF-κB signaling pathway in a degenerative intervertebral disc is gradually increased, regulating the over-expression of matrix-degrading enzymes.It plays an important role in the degradation of extra-cellular matrix.
10.3760/cma.j.issn.0376-2491.2017.17.011
Oxidative stress abrogates the degradation of KMT2D to promote degeneration in nucleus pulposus.
Xu Wenbin,Zhang Xuyang,Liu Gang,Zhu Mingjie,Wu Yizheng,Jie Zhiwei,Xie Ziang,Wang Shiyu,Ma Qingliang,Fan Shunwu,Fang Xiangqian
Biochimica et biophysica acta. Molecular basis of disease
Nucleus pulposus (NP) degeneration plays pivotal roles in intervertebral disc degeneration. The effect and mechanism of oxidative stress and epigenetics in NP degeneration is still unclear. We performed this study to evaluate the function of oxidative stress in NP and to explore the potential mechanism of ROS induced expression of matrix metalloproteinases (MMPs). We tested four methyltransferases, KMT2A, KMT2B, KMT2C and KMT2D in human NP samples, only KMT2D was significantly up-regulated in the severe degeneration samples. Knockdown of Kmt2d by siRNA significantly down-regulated the expression levels of catabolic enzymes including Mmp3, Mmp9 and Mmp13. Moreover, an interaction between KMT2D and ubiquitination was confirmed, and the application of HO abrogated this process. Co-IP assay confirmed that HO induced the phosphorylation of KMT2D to block the ubiquitination degradation, which was mainly mediated by phosphorylation of p38/MAPK. Further investigation suggested that ROS induced the alteration in levels of methylation is linked to H3K4me1 and H3K4me2, but not me3. However, usage of OICR-9429 (OICR) also suppressed the expression levels of Mmp3, Mmp9 and Mmp13. In an ex vivo model, application of OICR-9429 (OICR) also attenuated the degeneration of NP according to the H&E and Safranin-O/Fast Green staining assay, and the protein levels of MMP3, MMP9 and MMP13 were down-regulated, as well. In conclusion, we approved that oxidative stress induced ROS production promote the process of NP degeneration by enhancing KMT2D mediated transcriptional regulation of matrix degeneration related genes during NP degeneration.
10.1016/j.bbadis.2020.165888
Taurine attenuates ER stress‑associated apoptosis and catabolism in nucleus pulposus cells.
Yang Liuxie,Li Zhenhuan,Ouyang Yueping
Molecular medicine reports
Nucleus pulposus (NP) apoptosis and subsequent excessive degradation of the extracellular matrix (ECM) are key pathological characteristics of intervertebral disc degeneration (IDD). The present study aims to examine the signaling processes underlying the effects of taurine on IDD, with specific focus on endoplasmic reticulum (ER) stress‑mediated apoptosis and ECM degradation, in NP cells. To clarify the role of taurine in IDD, NP cells were treated with various concentrations of taurine and IL‑1β or thapsigargin (TG). Cell Counting Kit‑8, western blotting, TUNEL, immunofluorescence assays and reverse transcription‑quantitative PCR were applied to measure cell viability, the expression of ER stress‑associated proteins (GRP78, CHOP and caspase‑12), apoptosis and the levels of metabolic factors associated with ECM (MMP‑1, 3, 9, ADAMTS‑4, 5 and collagen II), respectively. Taurine was found to attenuate ER stress and prevent apoptosis in NP cells induced by IL‑1β treatment. Additionally, taurine significantly decreased the expression of ER stress‑activated glucose regulatory protein 78, C/EBP homologous protein and caspase‑12. TUNEL results revealed that taurine decreased the number of apoptotic TG‑treated NP cells. TG‑treated NP cells also exhibited characteristics of increased ECM degradation, supported by observations of increased MMP‑1, MMP‑3, MMP‑9 and A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)‑4 and ADAMTS‑5 expression in addition to decreased collagen‑II expression. However, taurine treatment significantly reversed all indicators of excessive ECM catabolism aforementioned. These data suggest that taurine can mediate protection against apoptosis and ECM degradation in NP cells by inhibiting ER stress, implicating therapeutic potential for the treatment of IDD.
10.3892/mmr.2022.12688
HtrA1 upregulates the expression of ADAMTS-5 in HNPCs via the ERK/NF-κB/JNK signaling pathway.
American journal of translational research
Intervertebral disc degeneration (IDD) is a form of chronic inflammation and is one of the most common disorders reported to be involved in low back pain (LBP). The pathophysiology of degeneration is not completely understood, but the consensus is that the degradation of extracellular matrix (ECM) proteins in the disc is the leading factor contributing to IDD. High temperature requirement A1 (HtrA1) is serine protease that has been shown to be increased in degenerated intervertebral discs as a result of an increase in the expression of matrix metalloproteinases (MMPs), but no study has focused on the effect of HtrA1 on a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTSs). In the present study, we successfully isolated human nucleus pulposus cells (HNPCs) from IDD patients who were our research subjects to elaborate on the potential role of HtrA1 in the pathogenesis of IDD. We confirmed that HtrA1 has the potential to induce the expression of ADAMTS-5 in a dose-dependent manner. Consistently, this was mediated by the ERK, NF-κB and JNK pathways. By using inhibitors of these pathways, the increase in ADAMTS-5 could be reduced. Our findings indicated that HtrA1 can induce the expression of ADAMTS-5 in HNPCs via the ERK/NF-κB/JNK signaling pathway, and our study also elucidated the involved induction mechanisms in HNPCs, which may provide new insights for the treatment of IDD.
Caspase 3 silencing inhibits biomechanical overload-induced intervertebral disk degeneration.
Yamada Katsuhisa,Sudo Hideki,Iwasaki Koji,Sasaki Naoki,Higashi Hideaki,Kameda Yusuke,Ito Manabu,Takahata Masahiko,Abumi Kuniyoshi,Minami Akio,Iwasaki Norimasa
The American journal of pathology
Intervertebral disk (IVD) degeneration causes debilitating low back pain in much of the worldwide population. No efficient treatment exists because of an unclear pathogenesis. One characteristic event early in such degeneration is the apoptosis of nucleus pulposus (NP) cells embedded in IVDs. Excessive biomechanical loading may also be a major etiology of IVD degeneration. The present study used in vitro and in vivo models of compressive loading to elucidate the underlying mechanism of IVD degeneration. In addition, we investigated whether the inhibition of apoptosis is a potential clinical therapeutic strategy for the treatment of IVD degeneration induced by biomechanical stress. A TUNEL assay showed that NP cell-agarose three-dimensional composite cultures subjected to uniaxial, unconfined, static, compressive loading exhibited a time-dependent increase in apoptosis. Western blot analysis revealed the up-regulation of several extracellular matrix-degrading enzymes and down-regulation of tissue inhibitor of metalloproteinase 1. These responses to compressive loading were all significantly inhibited by caspase 3 siRNA. In the in vivo model of compressive loading-induced IVD degeneration, a single local injection of caspase 3 siRNA significantly inhibited IVD degeneration by magnetic resonance imaging, histological findings, IHC, and TUNEL assay. The present study suggests that caspase 3 siRNA attenuates overload-induced IVD degeneration by inhibiting NP cell apoptosis and the expression of matrix-degrading enzymes.
10.1016/j.ajpath.2013.11.010
Fluid-induced, shear stress-regulated extracellular matrix and matrix metalloproteinase genes expression on human annulus fibrosus cells.
Chou Po-Hsin,Wang Shih-Tien,Yen Meng-Hua,Liu Chien-Lin,Chang Ming-Chau,Lee Oscar Kuang-Sheng
Stem cell research & therapy
BACKGROUND:Mechanical loading plays an important role in the regulation of extracellular matrix (ECM) homeostasis as well as pathogenesis of intervertebral disc (IVD) degeneration. The human annulus fibrosus (hAF) in the IVD is subjected to contact shear stress during body motion. However, the effects of shear stress on hAF cells remain unclear. This aim of the study was to investigate the expression of the ECM (COLI, COLIII and aggrecan) and matrix metalloproteinase (MMP-1, MMP-3 and ADAMTS-4) genes in hAF cells following fluid-induced shear stress in a custom-fabricated bio-microfluidic device. METHODS:hAF cells were harvested from degenerated disc tissues in routine spine surgery, staged by magnetic resonance imaging, expanded in monolayers and then seeded onto the bio-microfluidic device. The experimental groups were subjected to 1 and 10 dyne/cm(2) shear stress for 4 h, and no shear stress was applied to the control group. We used real time polymerase chain reaction for gene expression. RESULTS:Shear stress of 1 dyne/cm(2) exerted an anabolic effect on COLI and COLIII genes and catabolic effects on the aggrecan gene, while 10 dyne/cm(2) had an anabolic effect on the COLI gene and a catabolic effect on COLIII and aggrecan genes. The COLI gene was upregulated in a stress-dependent manner. Expression of MMP-1 was significantly higher in the 10 dyne/cm(2) group compared to the control group (P < 0.05), but was similar in the control and 1 dyne/cm(2) groups. Expression of MMP-3 and ADAMTS-4 were similar in all three groups. CONCLUSION:Taken together, hAF cells responded to shear stress. The findings help us understand and clarify the effects of shear stress on IVD degeneration as well as the development of a new therapeutic strategy for IVD degeneration.
10.1186/s13287-016-0292-5
Immunolocalization of MMP-19 in the human intervertebral disc: implications for disc aging and degeneration.
Gruber H E,Ingram J A,Hanley E N
Biotechnic & histochemistry : official publication of the Biological Stain Commission
Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix of the disc, but the presence of MMP-19 has not been explored. In other tissues, MMP-19 is known to act in proteolysis of the insulin-like growth factor (IGF) binding protein-3, thereby exposing this protein to make it available to influence cell behavior. MMP-19 also has been shown to inhibit capillary-like formation and thus play a role in the avascular nature of the disc. Using immunohistochemistry, normal discs from six subjects aged newborn through 10 years and 20 disc specimens from control donors or surgical patients aged 15-76 (mean age 40.2 years) were examined for immunolocalization of MMP-19; six Thompson grade I discs, five Thompson grade II, eight Thompson grade III, five Thompson grade IV, and one Thompson grade V discs were analyzed. The results indicate that in discs from young subjects, MMP-19 was uniformly localized in the outer annulus. In discs from adult donors and surgical patients, outer and inner annulus cells only occasionally showed MMP-19 localization. The greatest expression of MMP-19 was observed in young discs, and little expression was seen in older or degenerating discs. Because MMP-19 has been shown to regulate IGF-mediated proliferation in other tissues, its decline in the aging/degenerating disc may contribute to the age-related decrease in disc cell numbers.
10.1080/10520290500387607
[Expression of hypoxia-inducible factor-1alpha and matrix metalloproteinase-2 in degenerative lumbar intervertebral disc].
Wu Wei-ping,Jiang Jian-ming,Qu Dong-bin,Wei Qing-zhu,Jiang Hui
Nan fang yi ke da xue xue bao = Journal of Southern Medical University
OBJECTIVE:To investigate the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and matrix metalloproteinase-2 (MMP-2) in degenerative human intervertebral disc. METHODS:Specimens of the nucleus pulposus of the degenerative discs were obtained from patients with lumbar disc herniation (experimental group), and those from young patients suffering thoracolumbal vertebral bursting fracture served as the control. The experimental group was divided into three subgroups according to the magnetic resonance imaging (MRI) results. The tissue specimens from all the groups were examined for HIF-1alpha and MMP-2 expressions using immunohistochemistry. RESULTS:The tissues in the experimental group showed significantly higher expressions of HIF-1alpha and MMP-2 than those in the control group (P<0.05), and the expressions increased significantly with the severity of the degenerative changes of the intervertebral discs (P<0.05). A positive correlation was found between the expressions of the HIF-1alpha and MMP-2 in degenerative intervertebral discs. CONCLUSION:HIF-1alpha and MMP-2 may participate in the degeneration of human intervertebral discs, in which process their expressions show a common pattern of enhancement with the progression of the condition.
Fibrotic alterations in human annulus fibrosus correlate with progression of intervertebral disc herniation.
Castro A L,Ribeiro-Machado C,Oliveira C M,Teixeira G Q,Neidlinger-Wilke C,Pereira P,Vaz R,Barbosa M A,Gonçalves R M
Arthritis research & therapy
BACKGROUND:Intervertebral disc (IVD) herniation is characterized by annulus fibrosus failure (AF) in containing the nucleus pulposus (NP). IVD herniation involves cellular and extracellular matrix (ECM) alterations that have been associated with tissue fibrosis, although still poorly investigated. METHODS:Here, fibrotic alterations in human AF were evaluated, by characterizing the herniated ECM. Human AF samples (herniated lumbar IVD (n = 39, age 24-83) and scoliosis controls (n = 6, age 15-21)) were processed for transmission electron microscopy and histological/immunohistochemical analysis of fibrotic markers. Correlations between the fibrotic markers in AF ECM and the degree of NP containment (protused, contained and uncontained) and patients' age were conducted. RESULTS:Our results demonstrate that with herniation progression, i.e. loss of NP containment, human AF presents less stained area of sulphated glycosaminoglycans and collagen I, being collagen I fibres thinner and disorganized. On the other hand, fibronectin stained area and percentage of α-smooth muscle actin+ cells increase in human AF, while matrix metalloproteinase-12 (MMP12) production and percentage of macrophages (CD68+ cells) remain constant. These structural and biochemical fibrotic alterations observed in human AF with herniation progression occur independently of the age. CONCLUSIONS:The characterization of human AF here conducted evidence the presence of fibrosis in degenerated IVD, while highlighting the importance of considering the herniation progression stage, despite the patients' age, for a better understanding of the mechanisms behind AF failure and IVD herniation.
10.1186/s13075-021-02690-w
Human disc degeneration is associated with increased MMP 7 expression.
Le Maitre C L,Freemont A J,Hoyland J A
Biotechnic & histochemistry : official publication of the Biological Stain Commission
During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.
10.1080/10520290601005298
Resveratrol could reverse the expression of SIRT1 and MMP-1 in vitro.
Wu J W,Wang J J,Chen J B,Huang Y L,Wang H,Liu G H,Li L F,Kang M,Wang X G,Cai H H
Genetics and molecular research : GMR
Intervertebral disc degeneration is the main cause of lumbago disease, in which the extracellular matrix structure and moisture in the nucleus pulposus is lost continuously. In this study, we aimed to detect differential expression of silence mating type information regulation 2 homolog 1 (SIRT1) and matrix metalloproteinase-1 (MMP-1) in human intervertebral disc nucleus pulposus cells and to explore the effects of SIRT1 and MMP-1 on the development of the intervertebral disc degeneration. Intervertebral disc nucleus pulposus specimens from 41 patients who underwent lumbar protrusion resection at HuiZhou Municipal Central Hospital, during the period from October 2011 to December 2013, were studied in comparison with 23 control cases from patients who underwent fractured lumbar resection. In degenerated human intervertebral disc nucleus pulposus cells, the expression of SIRT1 is decreased and MMP-1 is increased compared with that of the control cells. Resveratrol could reverse these effects, thereby increasing the expression of SIRT1 (0.87 ± 0.07 vs 0.54 ± 0.04), Coll2α1 (0.90 ± 0.08 vs 0.38 ± 0.01), and aggrecan (0.69 ± 0.07 vs 0.42 ± 0.05) and decreasing the expression of MMP-1 (0.61 ± 0.03 vs 0.93 ± 0.08). These results suggest that resveratrol could possibly reverse the process of intervertebral disc degeneration and thus could be applied as a potential drug for the disease.
10.4238/2015.October.16.5
MiR-202-3p regulates interleukin-1β-induced expression of matrix metalloproteinase 1 in human nucleus pulposus.
Gene
MicroRNAs (miRNAs), small noncoding RNA molecules, have emerged as important factors during intervertebral disc degeneration. This study was to determine whether miR-202-3p regulates interleukin-1β (IL-1β)-induced expression of matrix metalloproteinase 1 (MMP-1) in human nucleus pulposus (NP) cells. Human NP cells were stimulated with IL-1β in vitro. MicroRNA arrays were used to determine the expression profile of 1971 human miRNAs and the miRNAs targets were identified using bioinformatics. In IL-1β-stimulated NP cells, 10 microRNAs were down-regulated, 2 microRNAs were up-regulated. There was a significant reduction in hsa-miR-202-3p (miR-202-3p) expression in the severe degenerative disc compared with mild degenerative disc. Down-regulation of miR-202-3p expression by IL-1β was correlated with up-regulation of MMP-1 expression in human NP cells. IL-1β-induced activation of MAP kinase (MAPK) and nuclear factor-κB (NF-κB) decreased miR-202-3p expression and induced MMP-1 expression. MiR-202-3p suppressed IL-1β-induced MMP-1 production. Conversely, treatment with anti-miR-202-3p remarkably increased MMP-1 production. In addition, mutation of the miR-202-3p binding site in the 3'-UTR of MMP-1 mRNA abolished miR-202-3p-mediated repression of reporter activity. Functional analysis showed that miR-202-3p could decrease type II collagen degradation, whereas overexpression of MMP-1 by Lentiviral-shMMP-1 abolished the effect of miR-202-3p on type II collagen degradation. These results suggest that miR-202-3p is an important regulator of MMP-1 in human nucleus pulposus and may contribute to the development of intervertebral disc degeneration.
10.1016/j.gene.2018.11.056
Intervertebral disk degeneration and herniation: the role of metalloproteinases and cytokines.
Grang L,Gaudin P,Trocme C,Phelip X,Morel F,Juvin R
Joint bone spine
This article reviews the role of metabolic factors, including metalloproteinases and cytokines, in the occurrence of degenerative disk disease and disk herniation. Given that mechanical factors alone cannot cause disk degeneration, studies must explore metabolic, genetic, nutritional, and age-related factors. Zinc metalloproteinases exert particularly important effects, not only directly, but also indirectly through promotion of neovascularization. The production of these enzymes is dependent on a number of cytokines and on the cell changes they induce. This complex effect acts both on disk matrix degeneration and on the pain generated by contact between the protruding disk and the nerve roots. However, it can have a favorable effect by promoting resorption of the herniated disk. Available data on the role for mechanical factors on the disk chondrocyte metabolism and on metalloproteinase production show that mechanical and metabolic factors interact closely to produce disk disorders.
10.1016/s1297-319x(01)00324-4
BRD4 inhibition regulates MAPK, NF-κB signals, and autophagy to suppress MMP-13 expression in diabetic intervertebral disc degeneration.
Wang Jianle,Hu Jianing,Chen Ximiao,Huang Chongan,Lin Jialiang,Shao Zhenxuan,Gu Mingbao,Wu Yaosen,Tian Naifeng,Gao Weiyang,Zhou Yifei,Wang Xiangyang,Zhang Xiaolei
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Diabetes mellitus may lead to intervertebral disc degeneration (IVDD). Matrix metalloproteinase-13 (MMP-13) is one of the major catabolic factors in extracellular matrix (ECM) metabolism of nucleus pulposus cells (NPCs) and contributes to diabetic IVDD. Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extraterminal protein family and is implicated in chronic inflammation. Here, we report that the expression of BRD4 and MMP-13 was elevated in diabetic nucleus pulposus tissues as well as in advanced glycation end products (AGEs)-treated NPCs; also, the regulatory effect of BRD4 on MMP-13 was studied. We found that MMP-13 was regulated by MAPK and NF-κB signaling as well as autophagy in AGEs-treated NPCs. Next, we explored the role of BRD4 in regulation of MAPK, NF-κB signaling, and autophagy. The results showed that BRD4 is the upstream regulator of all of these 3 factors, and inhibition of BRD4 may suppress MAPK and NF-κB signaling while activating autophagy in AGEs-treated NPCs. Finally, we demonstrated that BRD4 inhibition may suppress MMP-13 expression in diabetic NPCs as well as ; meanwhile, it may preserve ECM in diabetic rats. Our study demonstrates that inhibition of BRD4 may suppress MAPK and NF-κB signaling and activate autophagy to suppress MMP-13 expression in diabetic IVDD, and diabetic IVDD may be compromised by BRD4 inhibitors.-Wang, J., Hu, J., Chen, X., Huang, C., Lin, J., Shao, Z., Gu, M., Wu, Y., Tian, N., Gao, W., Zhou, Y., Wang, X., Zhang, X. BRD4 inhibition regulates MAPK, NF-κB signals, and autophagy to suppress MMP-13 expression in diabetic intervertebral disc degeneration.
10.1096/fj.201900703R
Long non-coding RNA PART1 promotes intervertebral disc degeneration through regulating the miR‑93/MMP2 pathway in nucleus pulposus cells.
Gao Dongmei,Hao Long,Zhao Zilong
International journal of molecular medicine
The aim of the present study was to investigate the role of the long non‑coding (lnc)RNA PART1 in nucleus pulposus (NP) cells derived from patients with intervertebral disc degeneration (IDD). The level of PART1 in degenerative NP tissues from patients with IDD, bulging and herniated discs was measured by reverse transcription‑quantitative PCR (RT‑qPCR) analysis. NP cells were isolated from patients with IDD and transfected with siPART1, after which time the growth ability of the NP cells was evaluated by Cell Counting Kit‑8 and colony formation assays, and cell apoptosis was measured by flow cytometry. The levels of the cell proliferation marker Ki‑67 and the apoptosis marker cleaved caspase‑3, and the levels of genes related to extracellular matrix (ECM) synthesis and degradation, were also evaluated by western blotting and RT‑qPCR, as appropriate. Bioinformatics methods predicted that miR‑93 was sponged by PART1, and matrix metallopeptidase (MMP)2 was targeted by miR‑93, which was further confirmed by dual‑luciferase reporter assay. The levels of miR‑93 and MMP2 were also measured in NP tissues, and further rescue experiments were performed to confirm the role of the PART1/miR‑93/MMP2 pathway in NP cells. PART1 was found to be upregulated in degenerative NP tissues, and siPART1 caused an increase in cell growth ability and ECM synthesis, whereas it decreased cell apoptosis and ECM degradation in NP cells. miR‑93 was downregulated and MMP2 was upregulated in degenerative NP tissues. Rescue experiments indicated that the effects of miR‑93 inhibitor on NP cells were abolished by siPART1, and the effect of miR‑93 mimic on NP cells was rescued by MMP2 overexpression. Thus, the results of the present study demonstrated that PART1 may regulate NP cell degeneration through the miR‑93/MMP2 pathway. These findings indicate a novel signaling axis in NP cells that may be explored for the treatment of IDD.
10.3892/ijmm.2020.4580
Correlation of matrix metalloproteinase-3 expression with patient age, magnetic resonance imaging and histopathological grade in lumbar disc degeneration.
Canbay Suat,Turhan Nesrin,Bozkurt Melih,Arda Kemal,Caglar Sukru
Turkish neurosurgery
AIM:The purpose of the present study is to analyze the expression of matrix metalloproteinase-3 (MMP-3), magnetic resonance imaging (MRI) grading and histopathological alterations of the intervertebral disc (IVD) for correlations with each other and with the age, gender and low back pain duration of the patients who had undergone operations for lumbar disc herniation (LDH). MATERIAL AND METHODS:Forty-two patients were admitted to our clinic with signs of LDH and underwent surgery for LDH at 48 IVD levels. In all cases, specimens for histological and immunohistochemical analyses were removed from the IVD space. Lumbar IVD degeneration on MRI of the 48 IVDs from which surgical specimens had been obtained was classified into five grades using the Pfirrmann classification. RESULTS:In the degenerated IVD, the expression of MMP-3, MRI grading and histopathological alterations of the IVD displayed significant correlation. Increased age is closely related with aforementioned alterations. There was no correlation between MMP-3 expression and age, gender and duration of the pain. CONCLUSION:For evaluating and treating IVD degeneration, MRI is a good and non-invasive diagnostic tool to determine the severity of degeneration. MMP-3 may be a therapeutic target of the degenerated IVD.
10.5137/1019-5149.JTN.7459-12.0
Heme oxygenase-1 attenuates IL-1β induced alteration of anabolic and catabolic activities in intervertebral disc degeneration.
Hu Bo,Shi Changgui,Xu Chen,Cao Peng,Tian Ye,Zhang Ying,Deng Lianfu,Chen Huajiang,Yuan Wen
Scientific reports
Intervertebral disc degeneration (IDD) is characterized by disordered extracellular matrix (ECM) metabolism, implicating subdued anabolism and enhanced catabolic activities in the nucleus pulposus (NP) of discs. Pro-inflammatory cytokines such as interleukin-1β (IL-1β) are considered to be potent mediators of ECM breakdown. Hemeoxygenase-1 (HO-1) has been reported to participate in cellular anti-inflammatory processes. The purpose of this study was to investigate HO-1 modulation of ECM metabolism in human NP cells under IL-1β stimulation. Our results revealed that expression of HO-1 decreased considerably during IDD progression. Induction of HO-1 by cobalt protoporphyrin IX attenuated the inhibition of sulfate glycosaminoglycan and collagen type II (COL-II) synthesis and ameliorated the reduced expressions of aggrecan, COL-II, SOX-6 and SOX-9 mediated by IL-1β. Induction of HO-1 also reversed the effect of IL-1β on expression of the catabolic markers matrix metalloproteinases-1, 3, 9 and 13. This was combined with inhibition of the activation of mitogen-activated protein kinase signaling. These findings suggest that HO-1 might play a pivotal role in IDD, and that manipulating HO-1 expression might mitigate the impairment of ECM metabolism in NP, thus potentially offering a novel therapeutic approach to the treatment of IDD.
10.1038/srep21190
Effects of nuclear factor kappa B signaling pathway in human intervertebral disc degeneration.
Zhongyi Sun,Sai Zhao,Chao Liu,Jiwei Tian
Spine
STUDY DESIGN:IL-1β (interleukin-1β) can activate human nucleus pulposus cells with or without nuclear factor kappa B (NF-κB) inhibition. We undertook a descriptive and mechanistic investigation of catabolic effects of NF-κB signaling pathway in intervertebral disc degenerative changes. OBJECTIVE:To clarify the mediatory role of NF-κB signaling pathway in human intervertebral disc degeneration (IDD). SUMMARY OF BACKGROUND DATA:IDD is a major cause of lower back pain, but the molecular mechanism behind this process is poorly understood. NF-κB is a family of transcription factors that play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic activation of NF-κB in many degenerative diseases, but its role in IDD has not been adequately explored. METHODS:Human nucleus pulposus cells in monolayer culture were exposed to IL-1β, which increases matrix-degrading enzyme activity in the nucleus pulposus, with or without NF-κB inhibition by BAY11-7082; ribonucleic acid was isolated for real-time polymerase chain reaction analysis of gene expression, Western blot analysis was performed to detect the changes of protein expression. RESULTS:NF-κB specific inhibitor BAY11-7082 significantly inhibited IL-1β-induced NF-κB activation. IL-1β-dependent gene upregulation of matrix metalloproteinase (MMP)-3, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5 was significantly reduced by NF-κB inhibition. The decreased gene expression of aggrecan and type II collagen, induced by IL-1β was also reversed by BAY11-7082. NF-κB inhibition reversed the IL-1β-induced changes of protein expression of MMP-3, MMP-9, MMP-13, ADAMTS-4, ADAMTS-5, aggrecan, and type II collagen. CONCLUSION:These findings demonstrate that the NF-κB signaling pathway is a key mediator of IDD and represents a therapeutic target for mitigating disc degenerative diseases. LEVEL OF EVIDENCE:N/A.
10.1097/BRS.0000000000000733
Importance of Metalloproteinase Enzyme Group in Selected Skeletal System Diseases.
International journal of molecular sciences
Bone tissue is a dynamic structure that is involved in maintaining the homeostasis of the body due to its multidirectional functions, such as its protective, endocrine, or immunological role. Specialized cells and the extracellular matrix (ECM) are responsible for the remodeling of specific bone structures, which alters the biomechanical properties of the tissue. Imbalances in bone-forming elements lead to the formation and progression of bone diseases. The most important family of enzymes responsible for bone ECM remodeling are matrix metalloproteinases (MMPs)-enzymes physiologically present in the body's tissues and cells. The activity of MMPs is maintained in a state of balance; disruption of their activity is associated with the progression of many groups of diseases, including those of the skeletal system. This review summarizes the current understanding of the role of MMPs in bone physiology and the pathophysiology of bone tissue and describes their role in specific skeletal disorders. Additionally, this work collects data on the potential of MMPs as bio-markers for specific skeletal diseases.
10.3390/ijms242417139
Intervertebral disc and endplate cells response to IL-1β inflammatory cell priming and identification of molecular targets of tissue degeneration.
De Luca P,de Girolamo L,Kouroupis D,Castagnetta M,Perucca Orfei C,Coviello D,Coco S,Correa D,Brayda-Bruno M,Colombini A
European cells & materials
Inflammation represents an important factor leading to metabolic imbalance within the intervertebral disc (IVD), conducive to degenerative changes. Therefore, a thorough knowledge of the IVD and endplate (EP) cell behaviour in such pathological environments is essential when designing regenerative therapeutic strategies. The present study aimed at assessing the molecular response of the IVD constitutive nucleus pulposus (NPCs)-, annulus fibrosus (AFCs)- and endplate (EPCs)-derived cells to interleukin (IL)-1β treatment, through large-scale, high-throughput microarray and protein analysis, identifying the differentially expressed genes and released proteins. Overall, the inflammatory stimulus downregulated stemness genes while upregulating pro-inflammatory, pro-angiogenic and catabolic genes, including matrix metalloproteases, which were not balanced by a concomitant upregulation of their inhibitors. Upregulation of anti-inflammatory and anabolic tumour necrosis factor inducible gene 6 protein (TNFAIP6), of IL-1 receptor antagonist (IL-1Ra) (at gene and protein levels) and of trophic insulin-like growth factor 1 (IGF1) was also observed in all cell types; IGF1 particularly in AFCs. An overall inhibitory effect of tumour necrosis factor alpha (TNFα) signal was observed in all cell types; however, EPCs showed the strongest anti-inflammatory behaviour. AFCs and EPCs shared the ability to limit the activation of the signalling mediated by specific chemokines. AFCs showed a slightly senescent attitude, with a downregulation of genes related to DNA repair or pro-mitosis. Results allowed for the identification of specific molecular targets in IVD and EP cells that respond to an inflammatory environment. Such targets can be either silenced (when pathological targets) or stimulated to counteract the inflammation.
10.22203/eCM.v039a15
Inhibited microRNA-494-5p promotes proliferation and suppresses senescence of nucleus pulposus cells in mice with intervertebral disc degeneration by elevating TIMP3.
Chen Gang,Zhou Xiaopeng,Li Hao,Xu Zhengkuan
Cell cycle (Georgetown, Tex.)
It has been unraveled that microRNAs (miRNAs) played crucial roles in processes of human diseases, while the role of miR-494-5p in intervertebral disc degeneration (IDD) remains scarcely studied. We aimed to investigate the mechanisms of miR-494-5p in IDD with the involvement of tissue inhibitor of metalloproteinase 3 (TIMP3). Expression of miR-494-5p and TIMP3 in IDD clinical specimens was assessed. The IDD models were established by needle punching, which were then injected with low expression of miR-494-5p or TIMP3 overexpression lentivirus to observe their effects on pathology and apoptosis in IDD mice. The nucleus pulposus cells were isolated and, respectively, treated with miR-494-5p inhibitor or TIMP3 overexpression plasmid to determine the viability, apoptosis, and senescence . Furthermore, the expression of Aggrecan, Col-2, Caveolin-1, and SA-β-gal in nucleus pulposus cells were measured. The target relation between miR-494-5p and TIMP3 was determined. An increased expression of miR-494-5p and a decreased expression of TIMP3 were found in IDD. Downregulation of miR-494-5p or overexpression of TIMP3 could relieve pathology and suppress nucleus pulposus cell apoptosis in IDD mice, as well as promote the viability and attenuate the apoptosis and senescence of nucleus pulposus cells from IDD mice. Moreover, inhibition of miR-494-5p or overexpression of TIMP3 upregulated Aggrecan and Col-2 expression while downregulated Caveolin-1 and SA-β-gal expression, and TIMP3 was the target gene of miR-494-5p. Results of this study indicated that the degradation of miR-494-5p ameliorates the development of IDD by elevating TIPM3, which may provide new targets for IDD treatment.
10.1080/15384101.2020.1843812
The Role of Hypoxia in Angiogenesis and Extracellular Matrix Regulation of Intervertebral Disc Cells During Inflammatory Reactions.
Kwon Woo-Keun,Moon Hong Joo,Kwon Taek-Hyun,Park Youn-Kwan,Kim Joo Han
Neurosurgery
BACKGROUND:The intervertebral disc (IVD) is an avascular structure, and is therefore stable under hypoxic conditions. Previous studies have demonstrated that hypoxia might be related to symptomatic degenerative disc diseases (DDDs); however, the pathomechanism is still poorly understood. OBJECTIVE:To identify the effect of hypoxia on the production of inflammatory mediators, angiogenic factors, and extracellular matrix-regulating enzymes of IVD cells during inflammatory reactions. METHODS:Human nucleus pulposus (NP) and annulus fibrosus (AF) cells harvested during surgery for DDDs were cultured in macrophage conditioned media or interleukin (IL)-1β-stimulated media under hypoxic (2%) and normoxic (21%) conditions. Hypoxia-inducible factor-1α transcription factor activation was analyzed by western blotting. IL-6, IL-8, vascular endothelial growth factor (VEGF), vascular cell adhesion molecule (VCAM), matrix metalloproteinase (MMP)-1, MMP-3, tissue inhibitor of metalloprotease (TIMP)-1, and TIMP-2 in conditioned media were measured by an enzyme-linked immunosorbent assay. RESULTS:NP cells expressed higher hypoxia-inducible factor-1α in the IL-1β-stimulated group under hypoxic condition. MMP-1 was significantly increased in the AF cells under hypoxic condition; TIMP-1 and TIMP-2 were significantly decreased in both naïve NP and AF cells during hypoxia. Both cells in macrophage conditioned media significantly diminished the production of IL-6 and VCAM, while VEGF significantly increased during hypoxia. After 1 ng/mL IL-1β stimulation, IL-8, VEGF, MMP-1, and MMP-3 were significantly increased in both cell types during hypoxia, while VCAM, TIMP-1, and TIMP-2 were decreased. CONCLUSION:We found that hypoxia can enhance the angiogenic ability of IVD during inflammatory reactions, and cause progress in development of DDD via extracellular matrix regulation in this in vitro study.
10.1093/neuros/nyx149
Degenerate intervertebral disc-like pH induces a catabolic mechanoresponse in human nucleus pulposus cells.
JOR spine
Mechanical stimulation is known to influence intervertebral disc (IVD) cell behavior and function, but the effect on disc cells is routinely considered in isolation from other microenvironmental factors. Acidic pH has been shown to be a prominent and detrimental microenvironmental factor present in degenerate IVDs, but its influence on the human disc cell mechanoresponse has never been studied. We investigated the response of agarose-encapsulated human nucleus pulposus (NP) cells to 0.004 MPa, 1.0 Hz and 1 hour of compression (Flexcell FX4000 Compression System) under pH conditions representative of nondegenerate (pH 7.1) and degenerate (pH 6.5) IVDs. Cell viability, extracellular matrix production, and expression of anabolic/anti-catabolic and catabolic genes were assessed. We report that preculture of NP cells in agarose gels was required in order for cells to be mechanoresponsive, and this correlated with increased type VI collagen, α5β1 integrin, and fibronectin expression. Furthermore, the matrix homeostatic response observed at pH 7.1 (representative of nondegenerate IVDs; increased aggrecan [AGC], tissue inhibitor of metalloproteinases-1 [TIMP1], matrix metalloproteinase-3 [MMP3], a disintegrin and metalloproteinase with thrombospondin motif-5 [ADAMTS5] gene expression) was RGD-integrin dependent, whereas only MMP3 remained mechanoresponsive at pH 6.5, and this was independent of RGD-integrins. Our findings suggest differential mechanotransduction pathways operating for specific genes, with RGD-integrin dependent AGC expression, but not RGD-independent MMP3 expression, inhibited at pH representative of degenerate IVDs (pH 6.5), which could contribute to the catabolic phenotype observed during IVD degeneration. CLINICAL SIGNIFICANCE:Characterizing the influence of the mechanical and chemical intervertebral disc microenvironment on disc cells, particularly in disc degeneration, could help develop future therapeutic strategies for the treatment of discogenic back pain.
10.1002/jsp2.1004
Bu-Shen-Huo-Xue-Fang modulates nucleus pulposus cell proliferation and extracellular matrix remodeling in intervertebral disk degeneration through miR-483 regulation of Wnt pathway.
Yang Shaofeng,Li Linghui,Zhu Liguo,Zhang Chao,Li Zhaoyong,Guo Yantao,Nie Ying,Luo Zhenhua
Journal of cellular biochemistry
Intervertebral disk degeneration (IDD) has been widely considered as one of the main causes for low back pain, which can cause a severe impact to human health and huge economic burden to worldwide society. IDD pathogenesis can be affected by extensive degradation of extracellular matrix (ECM) and the hyperproliferation of nucleus pulposus (NP) cells. During the IDD process, expression of the ECM degradation enzymes matrix metalloproteinase and ADAMTS increases, whereas expression of ECM synthesis-related aggrecan and COL2A1 decreases. In addition, the Wnt signaling pathway is reportedly involved in the process of IDD. Bu-Shen-Huo-Xue-Fang (BSHXF), a Chinese traditional medicine formula that contains six Chinese traditional medicinal herbs, is widely used in the treatment of IDD. Herein, we obtained the serum containing BSHXF from BSHXF-fed rat and demonstrated that the BSHXF promoted NP cell proliferation and ECM synthesis through the Wnt signaling pathway. By using DIANA online tools and luciferase reporter gene assays, we confirmed that miR-483-3p and miR-23c regulated CTNNB1 and GSK3B, respectively, through direct targeting, thereby affecting the effect of BSHXF on NP cell proliferation and ECM synthesis through the Wnt signaling pathway. Taken together, we demonstrated the function and mechanism of BSHXF in regulating NP cell proliferation and ECM remodeling through the Wnt signaling pathway during IDD.
10.1002/jcb.26760
Alterations in biochemical components of extracellular matrix in intervertebral disc herniation: role of MMP-2 and TIMP-2 in type II collagen loss.
Kozaci Leyla Didem,Guner Alev,Oktay Gulgun,Guner Gul
Cell biochemistry and function
Alterations in the composition of intervertebral disc extracellular matrix, mainly collagen and proteoglycans, may cause changes in mechanical properties of the disc, leading to dysfunction, nerve root compression, and herniation with severe clinical manifestations. Matrix metalloproteinases may be involved in degradation by hydrolysing extracellular matrix components. Inhibitors of matrix metalloproteinases, in contrast, function in the maintenance of degradation control. In this study, we investigated: (i) whether the level of matrix degradation correlated with the duration of the symptomatic disease, (ii) roles of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) in intervertebral disc degeneration. Nucleus pulposus of intervertebral discs were obtained from 22 patients and analysed for collagen and proteoglycan contents, and pro-MMP-2, TIMP-2 levels. Collagen content was determined as hydroxyproline and proteoglycan content was measured as glycosaminoglycans. The loss in matrix components did not correlate with the duration of the degenerative disc disease. Pro-MMP-2 levels were higher at early stages of the degenerative disc disease (r = -0.495, P < 0.05). TIMP-2 levels were similar in all samples. Pro-MMP-2 and TIMP-2 levels negatively correlated in herniated discs samples (r = -0.855, P < 0.01). Pro- MMP-2 levels negatively correlated with the collagen content in herniated disc material. Our findings may suggest a silent period of active disease prior to symptomatic outcome during which irreversible matrix loss occurs. Involvement of other proteolytic enzymes at different stages of the disease should also be investigated to help to control the degradation cascade at relatively early stages of disc degeneration before the clinical onset of disease.
10.1002/cbf.1250
Construction of a Potentially Functional circRNA-miRNA-mRNA Network in Intervertebral Disc Degeneration by Bioinformatics Analysis.
BioMed research international
BACKGROUND:The competing endogenous RNA- (ceRNA-) mediated regulatory mechanisms are known to play a pivotal role in intervertebral disc degeneration (IDD). Our research intended to establish a ceRNA regulatory network related to IDD through bioinformatics analyses. METHODS:The expression profiles of circRNA, miRNA, and mRNA were obtained from the public Gene Expression Omnibus (GEO) datasets. Then, we use sequence-based bioinformatics methods to select differentially expressed mRNAs (DEmRNAs), microRNAs (DEmiRNAs), or circRNAs (DEcircRNAs) related to IDD. We used ChEA3 to verify the targets of transcription factors (TFs). Then, we used DAVID to annotate the DEmRNAs. Finally, we constructed a potentially circRNA-miRNA-mRNA network related to IDD by predicting in the database (ENCORI, TargetScan, miRecords, miRmap, and circBank). RESULTS:We identified 31 common DEmRNAs by Venn analysis, of which MMP2 was regarded as the key hub genes. Simultaneously, miR-423-5p and miR-185-5p were predicted as the upstream molecules of MMP2. Furthermore, a total of six DEcircRNAs were predicted as the upstream circRNAs of miR-423-5p and miR-185-5p. Then, a potential circRNA-miRNA-mRNA network related to IDD was constructed by bioinformatics analysis. CONCLUSION:A comprehensive ceRNA regulatory network was constructed, which was found to be significant in IDD progression.
10.1155/2021/8352683
In vitro model of distinct catabolic and inflammatory response patterns of endothelial cells to intervertebral disc cell degeneration.
Hwang Min Ho,Son Hyeong-Guk,Kim Joohan,Choi Hyuk
Scientific reports
To evaluate dominant cell-to-cell paracrine interactions, including those of human annulus fibrosus (AF), nucleus pulposus (NP), and endothelial cells (ECs), in the production of inflammatory mediators and catabolic enzymes, ECs was cultured in soluble factors derived from AF or NP cells (AFCM or NPCM, respectively) and vice versa. We analysed IL-6 and -8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and -3, nerve growth factor (NGF)-β, and brain-derived neurotrophic factors (BDNFs) with qRT-PCR and ELISA. We implement a microfluidic platform to analyse migration properties of AF and NP cells and ECs in 3D cultures. Our results show that IL-1β-stimulated AF cells produced significantly higher levels of IL-6 and -8, VEGF, and MMP-1 than IL-1β-stimulated NP cells. However, production of IL-6 and -8, VEGF, and MMP-3 was significantly higher in NP cells than in AF cells, under the presence of ECs conditioned medium. We observed considerable migration of NP cells co-cultured with ECs through the microfluidic platform. These results suggest that AF cells may play a major role in the initial degeneration of intervertebral disc. Furthermore, it was found that interactions between NP cells and ECs may play a significant role in the development or progression of diseases.
10.1038/s41598-020-77785-6
Novel function of TWEAK in inducing intervertebral disc degeneration.
Wako Masanori,Haro Hirotaka,Ando Takashi,Hatsushika Kyosuke,Ohba Tetsuro,Iwabuchi Sadahiro,Nakao Atsuhito,Hamada Yoshiki
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
The goal of this research was to examine the role of TWEAK in normal disc cells and to investigate its potential role in disc degeneration. We performed histological examinations of disc tissues and assessed the role of the novel cytokine TWEAK using murine organ disc culture. The expression of both TWEAK and its receptor, Fn14, in discs was confirmed by immunohistochemistry and quantitative real-time PCR. TWEAK induced disc cells to generate MMP-3 in a dose- and time-dependent manner. This induction was strongly inhibited in the presence of a neutralizing antibody to TWEAK or a chimeric Fn14/Fc fusion protein. In disc tissues derived from TNF-alpha receptor 1- or TNF-alpha receptor 2-deficient mice, recombinant TWEAK modestly induced MMP-3. In contrast, in disc cultures lacking TWEAK, tissues from wild-type mice or receptor-deficient mice failed to express MMP-3. Furthermore, aggrecan expression was potently abrogated in a time-dependent manner in the presence of recombinant TWEAK. This is the first report to confirm expression of TWEAK and its receptor Fn14 in murine intervertebral disc tissues. The data suggest that TWEAK plays a role in MMP-3 up-regulation and aggrecan down-regulation in disc tissues, resulting in proteoglycan degradation and promotion of disc degeneration.
10.1002/jor.20445
Modified expression of the ADAMTS enzymes and tissue inhibitor of metalloproteinases 3 during human intervertebral disc degeneration.
Pockert Aneta J,Richardson Stephen M,Le Maitre Christine L,Lyon Malcolm,Deakin Jonathan A,Buttle David J,Freemont Anthony J,Hoyland Judith A
Arthritis and rheumatism
OBJECTIVE:Intervertebral disc degeneration is linked to loss of extracellular matrix (ECM), particularly the early loss of aggrecan. A group of metalloproteinases called aggrecanases are important mediators of aggrecan turnover. The present study was undertaken to investigate the expression of the recognized aggrecanases and their inhibitor, tissue inhibitor of metalloproteinases 3 (TIMP-3), in human intervertebral disc tissue. METHODS:Twenty-four nondegenerated and 30 degenerated disc samples were analyzed for absolute messenger RNA (mRNA) copy number of ADAMTS 1, 4, 5, 8, 9, and 15 and TIMP-3 by real-time reverse transcription-polymerase chain reaction. Thirty-six formalin-fixed embedded intervertebral disc samples of varying grades of degeneration were used for immunohistochemical analyses. In addition, samples from 8 subjects were analyzed for the presence of matrix metalloproteinase (MMP)- and aggrecanase-generated aggrecan products. RESULTS:Messenger RNA for all the aggrecanases other than ADAMTS-8 was identified in intervertebral disc tissue, as was mRNA for TIMP-3. Levels of mRNA expression of ADAMTS 1, 4, 5, and 15 were significantly increased in degenerated tissue compared with nondegenerated tissue. All these aggrecanases and TIMP-3 were also detected immunohistochemically in disc tissue, and numbers of nucleus pulposus cells staining positive for ADAMTS 4, 5, 9, and 15 were significantly increased in degenerated tissue compared with nondegenerated tissue. Aggrecan breakdown products generated by MMP and aggrecanase activities were also detected in intervertebral disc tissue. CONCLUSION:The aggrecanases ADAMTS 1, 4, 5, 9, and 15 may contribute to the changes occurring in the ECM during intervertebral disc degeneration. Targeting these enzymes may be a possible future therapeutic strategy for the prevention of intervertebral disc degeneration and its associated morbidity.
10.1002/art.24291
IL-21 Is Positively Associated with Intervertebral Disc Degeneration by Interaction with TNF-α Through the JAK-STAT Signaling Pathway.
Chen Bin,Liu Yi,Zhang YuanQiang,Li JingKun,Cheng KaiYuan,Cheng Lei
Inflammation
This study was conducted in order to investigate the function of IL-21 in intervertebral disc degeneration. The serum concentration of IL-21 in patients with lumbar disc herniation (LDH) was examined by ELISA. Immunohistochemistry and western blot analysis were performed to detect the expression of IL-21, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-7), and tumor necrosis factor alpha (TNF-α) in degenerated intervertebral disc (IVD) tissues of human and rat. Moreover, nucleus pulposus (NP) cells were treated with 0, 10, 100, and 1000 ng/mL of IL-21 cytokine with and without AG490. TNF-α, ADAMTS-7, and matrix metalloproteinases-13 (MMP-13) mRNA expression was determined by RT-PCR. The expression of signal transducers and activators of transcription, STAT-1, STAT-3, and STAT-5b, was detected by western blot. IL-21 concentration level is higher in the degenerated group and positively correlates with the visual analog score (VAS). IL-21, ADAMTS-7, and TNF-α can be detected in the degenerative NP tissues in both human and rat degenerated NP tissues. The mRNA expression of ADAMTS-7, TNF-α, and MMP-13 was enhanced after stimulation with IL-21. Compared to control, STAT-1, STAT-3, and STAT-5b expression was also enhanced after IL-21 treatment, with STAT-3 being the most significantly enhanced; furthermore, expression was significantly reduced after treatment with AG490. The mRNA expression of TNF-α was markedly reduced after treatment with AG490 compared to treatment with IL-21 only. IL-21 is involved in the pathological development of IVD degeneration and IL-21 could aggravate IVD degeneration by stimulating TNF-α through the STAT signaling pathway.
10.1007/s10753-017-0508-6
Whole-body vibration of mice induces progressive degeneration of intervertebral discs associated with increased expression of Il-1β and multiple matrix degrading enzymes.
McCann Matthew R,Veras Matthew A,Yeung Cynthia,Lalli Gurkeet,Patel Priya,Leitch Kristyn M,Holdsworth David W,Dixon S Jeffrey,Séguin Cheryle A
Osteoarthritis and cartilage
OBJECTIVE:Whole-body vibration (WBV) is a popular fitness trend based on claims of increased muscle mass, weight loss and reduced joint pain. Following its original implementation as a treatment to increase bone mass in patients with osteoporosis, WBV has been incorporated into clinical practice for musculoskeletal disorders, including back pain. However, our recent studies revealed damaging effects of WBV on joint health in a murine model. In this report, we examined potential mechanisms underlying disc degeneration following exposure of mice to WBV. METHODS:Ten-week-old male mice were exposed to WBV (45 Hz, 0.3 g peak acceleration, 30 min/day, 5 days/week) for 4 weeks, 8 weeks, or 4 weeks WBV followed by 4 weeks recovery. Micro-computed tomography (micro-CT), histological, and gene expression analyses were used to assess the effects of WBV on spinal tissues. RESULTS:Exposure of mice to 4 or 8 weeks of WBV did not alter total body composition or induce significant changes in vertebral bone density. On the other hand, WBV-induced intervertebral disc (IVD) degeneration, associated with decreased disc height and degenerative changes in the annulus fibrosus (AF) that did not recover within 4 weeks after cessation of WBV. Gene expression analysis showed that WBV for 8 weeks induced expression of Mmp3, Mmp13, and Adamts5 in IVD tissues, changes preceded by increased expression of Il-1β. CONCLUSIONS:Progressive IVD degeneration induced by WBV was associated with increased expression of Il-1β within the IVD that preceded Mmp and Adamts gene induction. Moreover, WBV-induced IVD degeneration is not reversed following cessation of vibration.
10.1016/j.joca.2017.01.004
Expression profiles of MMP-1 and TIMP-1 in lumbar intervertebral disc degeneration.
Deng B,Ren J Z,Meng X Q,Pang C G,Duan G Q,Zhang J X,Zou H,Yang H Z,Ji J J
Genetics and molecular research : GMR
Lumbar intervertebral disc degeneration (IDD) is a common clinical pathology and has become a focus for research in recent years. Matrix metalloproteinases (MMPs) are enzymes responsible for the degradation of almost all extracellular matrix proteins (ECM). The over-expression of MMPs or tissue inhibitors of metalloproteinases (TIMPs) may disrupt the dynamic balance of the ECM. Therefore, in the current study, the expression levels of MMP-1 and TIMP-1 in lumbar IDD patients were evaluated in an attempt to elucidate their role in IDD pathogenesis and progression. In total, 60 IDD patients were recruited as the experimental group, along with 20 cases of lumbar vertebral injury without disc degeneration as the control group. Preoperative venous blood samples were collected, and intervertebral disc tissues were collected from the lesion during surgery. Serum and tissue levels of MMP-1 and TIMP-1 were quantified by enzyme-linked immunosorbent assay and immunohistochemical staining, respectively. Serum and tissue MMP-1 levels in IDD patients were significantly higher than those in the control group (P < 0.05). Additionally, sub-group analysis revealed that severe IDD patients had higher MMP-1 levels compared with mild or moderate IDD patients (P < 0.05). However, there were no significant differences in TIMP- 1 levels in either the serum or tissues of IDD patients compared to patients in the control group (P > 0.05). These results demonstrate that MMP-1 expression is increased in IDD, with higher expression observed in more severe cases, whereas TIMP-1 expression was similarly expressed in both normal and degenerated discs.
10.4238/2015.December.29.16
Increased MMP-2 activity during intervertebral disc degeneration is correlated to MMP-14 levels.
Rutges J P H J,Kummer J A,Oner F C,Verbout A J,Castelein R J M,Roestenburg H J A,Dhert W J A,Creemers L B
The Journal of pathology
Intervertebral disc (IVD) degeneration is associated with the increased expression of several matrix metalloproteinases (MMPs), in particular MMP-2. However, little is known about the actual activity of MMP-2 in healthy and degenerated discs, or what mechanisms are involved in its activation. A major activation pathway involves complex formation with MMP-14 and a tissue inhibitor of metalloproteinases-2 (TIMP-2). In a series of 56 human IVDs, obtained at autopsy and graded according to the Thompson score (I-V), we analysed whether MMP-2 activity was increased in different stages of IVD degeneration and to what extent activation was related to the production of MMP-14 and TIMP-2. MMP-2 activation and production were quantified by gelatin zymography. Immunohistochemical staining of MMP-14 and TIMP-2 was quantified with a video overlay-based system. A positive correlation was observed between the amount of active MMP-2 and pro-MMP-2 and degeneration grade (p < 0.001, correlation coefficient (CC) 0.557; and p < 0.001, CC 0.556, respectively). MMP-2 activity correlated positively with MMP-14 and less strongly with TIMP-2 (p = 0.001, CC 0.436; and p = 0.03, CC 0.288, respectively). Moreover, immunopositivity for MMP-14 correlated to degeneration grade (p = 0.002, CC 0.398). IVD degeneration was associated with the activity of MMP-2 and the correlation of its activation with MMP-14 production suggests MMP-14 activates MMP-2 during degeneration. As MMP-14 is capable of activating several other enzymes that are also thought to be involved in IVD degeneration, it may be a key mediator of the degenerative process.
10.1002/path.2317
Ionizing Radiation Induces Disc Annulus Fibrosus Senescence and Matrix Catabolism via MMP-Mediated Pathways.
International journal of molecular sciences
Previous research has identified an association between external radiation and disc degeneration, but the mechanism was poorly understood. This study explores the effects of ionizing radiation (IR) on inducing cellular senescence of annulus fibrosus (AF) in cell culture and in an in vivo mouse model. Exposure of AF cell culture to 10-15 Gy IR for 5 min followed by 5 days of culture incubation resulted in almost complete senescence induction as evidenced by SA-βgal positive staining of cells and elevated mRNA expression of the p16 and p21 senescent markers. IR-induced senescent AF cells exhibited increased matrix catabolism, including elevated matrix metalloproteinase (MMP)-1 and -3 protein expression and aggrecanolysis. Analogous results were seen with whole body IR-exposed mice, demonstrating that genotoxic stress also drives disc cellular senescence and matrix catabolism in vivo. These results have important clinical implications in the potential adverse effects of ionizing radiation on spinal health.
10.3390/ijms23074014
ADAMTS-5 and intervertebral disc degeneration: the results of tissue immunohistochemistry and in vitro cell culture.
Zhao Chang-Qing,Zhang Yue-Hui,Jiang Sheng-Dan,Li Hai,Jiang Lei-Sheng,Dai Li-Yang
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Matrix metalloproteinases (MMPs) are known to be involved in IVD degeneration by hydrolyzing the extracellular matrix (ECM), especially the collagens. The degradation of proteoglycans, which is another main ECM component in the IVD, however, has not been extensively investigated. This study aimed to determine the expression of ADAMTS-5 in human herniated intervertebral disc (IVD) tissues and to investigate whether interleukin-1β (IL-1β)-induced expression of ADAMTS-5 is mediated by nitric oxide (NO). Forty-five herniated IVDs were harvested and immunostained to determine the distribution and type of ADAMTS-5 expressing cells. Rat NP cells maintained in alginate beads were treated with IL-1β, accumulation of NO was detected by Griess reaction, the expression of ADAMTS-5 and inducible nitric oxide synthase (iNOS) was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), the content of proteoglycans in alginate beads was visualized by alcian blue staining, and the effect of aminoguanidine on the changes in alginate beads induced by IL-1β treatment were also examined. Immunohistochemical results from 45 herniated discs showed that ADAMTS-5-positive cells are commonly seen in cell clusters, that the percentage of ADAMTS-5-positive cells was higher in uncontained herniated discs than in contained ones, and that the percentage of ADAMTS-5-positive cells correlated with the age of the patients. IL-1β treatment resulted in increased accumulation of NO, increased expression of ADAMTS-5 and iNOS, whereas the accumulation of proteoglycan in alginate beads decreased. Aminoguanidine significantly reversed the changes in alginate beads induced by IL-1β treatment. We thus suggested that ADAMTS-5 is probably involved in the process of IVD degeneration, and that IL-1β-induced expression of ADAMTS-5 is mediated by NO.
10.1002/jor.21285
Low expression of miR-142-3p promotes intervertebral disk degeneration.
Xue Jianmin,Hu Baoyang,Xing Wenhua,Li Feng,Huang Zhi,Zheng Wenkai,Wang Bo,Zhu Yong,Yang Xuejun
Journal of orthopaedic surgery and research
BACKGROUND:Intervertebral disk degeneration (IDD) is a degenerative disease characterized by cytoplasm loss and extracellular matrix degradation. Numerous evidence reported that miRNAs participated in IDD development. Nevertheless, the function of miR-142-3p in IDD development remains unknown. This study mainly explored the potential role and function of miR-142-3p in IDD development. METHODS:One percent fetal bovine serum was used to induce the degeneration of ATDC5 cells, and miR-142-3p level was examined by qRT-PCR. Then, miR-142-3p mimic/inhibitor and its corresponding negative control were transfected into ATDC5 normal and degenerative cells. Viability, migration, invasion, apoptosis, cycle, Bax, Bcl-2, P62, and Beclin1 expression levels were assessed using CCK8, wound healing assay, annexin V-FITC/PI staining, western blot, and qRT-PCR, respectively. RESULTS:The results revealed that the expression levels of MMP13, ADAMTS5, MMP3, and Col-X were increased as well as the expression levels of SOX-9 and Col-II were reduced in ATDC5 degenerative cells, indicating the degeneration model was constructed. We observed that miR-142-3p was decreased in ATDC5 degenerative cells and its suppression could promote ATDC5 cell degeneration. However, miR-142-3p overexpression could reverse the cell viability inhibition, as well as apoptosis and autophagy enhancement in ATDC5 degenerative cells. CONCLUSIONS:Our results proved that miR-142-3p may play an important role in disk degeneration. Further animal study is needed to illustrate the role of the miR-142-3p in IDD development.
10.1186/s13018-020-02194-4
Aquaporin 3 protects against lumbar intervertebral disc degeneration via the Wnt/β-catenin pathway.
Xie Huanxin,Jing Yongbin,Xia Jingjun,Wang Xintao,You Changcheng,Yan Jinglong
International journal of molecular medicine
Previous studies have demonstrated that the expression of aquaporin 3 (AQP3), a water channel which promotes glycerol permeability and water transport across cell membranes, is reduced in degenerative lumbar intervertebral disc (IVD) tissues. However, the role of AQP3 in the pathogenesis of IVD degeneration has not recieved much scholarly attention. The objective of the present study was to investigate the effect of AQP3 on cell proliferation and extracellular matrix (ECM) degradation in human nucleus pulposus cells (hNPCs) using gain-of-function and loss-of-function experiments, and to determine whether Wnt/β-catenin signaling is involved in the effect of AQP3 on IVD degeneration. hNPCs were transfected with the AQP3-pcDNA3.1 plasmid or AQP3 siRNA to overexpress or suppress AQP3. An MTT assay was performed to determine cell proliferation, and we found that AQP3 promoted hNPC proliferation. The expression of aggrecan, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4 and ADAMTS5 was detected using western blot analysis, to examine the effect of AQP3 on ECM degradation in hNPCs. The results revealed that AQP3 inhibited ECM degradation in hNPCs. In addition, we found that Wnt/β-catenin signaling was suppressed by AQP3. However, the effect of AQP3 on hNPC proliferation and ECM degradation was reversed by treatment with lithium chloride, a known activator of Wnt/β‑catenin signaling. In conclusion, using in vitro and in vivo tests, we have reported for the first time, to the best of our knowledge, that AQP3 exerts protective effects against IVD degeneration, and these are effected, at least partially, through the inhibition of Wnt/β-catenin signaling.
10.3892/ijmm.2016.2470
Correlation of matrix metalloproteinase (MMP)-1, -2, -3, and -9 expressions with demographic and radiological features in primary lumbar intervertebral disc disease.
Basaran Recep,Senol Mehmet,Ozkanli Seyma,Efendioglu Mustafa,Kaner Tuncay
Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia
Degeneration of IVD is a progressive and irreversible process and can be evaluated with immunohistochemical examination or radiological grading. MMPs are a family of proteolytic enzymes and involved in the degradation of the matrix components of the IVD. We aimed to compare MMP-1, -2, -3, and -9 expressions with demographic features, visual analogue scale (VAS), Oswestry Disability Index (ODI) and radiological (MRI) grades. The study involved 60 participants. We recorded data about age, complaint, radiological imaging, expression levels of MMP-1, -2, -3, and -9, ODI and VAS for back pain retrospectively. Intervertebral disc degeneration was graded on a 0-5 scale according to the Pfirrmann classification. As a result of the study, the median age was 52.09±12.74years. There were statistical significances between age and MMP-1, and MMP-2. There was a close correlation between grade and MMP-9. We found correlation between the VAS and the MMP-9 expression. In addition, there was relationship between expression of MMP-2 and MMP-1, MMP-3, MMP-9. In conclusion, the expressions of MMP-1 and -2 are increased with aging. There was no relationship between radiological evaluation of IVDD and aging. Increased expression of MMPs affected IVDD positively. The relationship with MMPs is not explained. This study adds to our understanding of the interaction between MMPs and IVDD.
10.1016/j.jocn.2017.03.001
Matrix metalloproteinase-3 and vitamin d receptor genetic polymorphisms, and their interactions with occupational exposure in lumbar disc degeneration.
Yuan Han-Yan,Tang Ying,Liang You-Xin,Lei Ling,Xiao Guo-Bing,Wang Sheng,Xia Zhao-Lin
Journal of occupational health
OBJECTIVES:To investigate the occupational and genetic risk factors inducing lumbar disc degeneration in a Chinese population, and to explore their synergistic interactions. METHODS:A case-control study involving 178 low back pain patients with lumbar disc degeneration and 284 controls was carried out. Five types of work-related factors were investigated using questionnaires. Polymerase chain reaction and restriction fragments length polymorphism was used to detect the polymorphisms of MMP-3 (matrix metalloproteinase-3)(rs731236), VDR-Taq (vitamin D receptor-Taq) and VDR-Apa (vitamin D receptor-Apa)(rs35068180). Rothman's synergy index was used to measure the synergistic interactions between gene polymorphisms and occupational risk factors. RESULTS:Family history of lumbar disc diseases, back injury history, whole-body vibration, bending/twisting, heavy physical workload, age, mutation alleles 5A of MMP-3 and A of VDR-Apa were significantly associated with lumbar disc degeneration (OR=12.70, 11.79, 8.96, 5.46, 1.05, 1.96 and 1.70, respectively, p<0.05). Synergistic interactions existed between the mutation allele 5A of MMP-3 and whole-body vibration exposure, the mutation allele 5A of MMP-3 and bending/twisting, and the mutation allele A of VDR-Apa and bending/twisting (SI=13.27, 2.91, 2.35, respectively, p<0.05). CONCLUSIONS:The results of this study suggest that gene-occupation interaction might play a certain role in exaggerating lumbar disc degeneration. There is a possibility that subjects who carry mutation alleles 5A of MMP-3 and/or A of VDR-Apa are more vulnerable to lumbar disc degeneration when they are exposed to whole-body vibration and/or bending/twisting under ergonomic loads.
10.1539/joh.l8149
Mitochondrial-derived reactive oxygen species (ROS) play a causal role in aging-related intervertebral disc degeneration.
Nasto Luigi A,Robinson Andria R,Ngo Kevin,Clauson Cheryl L,Dong Qing,St Croix Claudette,Sowa Gwendolyn,Pola Enrico,Robbins Paul D,Kang James,Niedernhofer Laura J,Wipf Peter,Vo Nam V
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Oxidative damage is a well-established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O(2)) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O(2)). Human disc cells grown at 20% O(2) showed increased levels of mitochondrial-derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria-targeted reactive oxygen species (ROS) scavenger XJB-5-131 blunted the adverse effects caused by 20% O(2). Importantly, we demonstrated that treatment of accelerated aging Ercc1(-/Δ) mice, previously established to be a useful in vivo model to study age-related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial-derived ROS contributes to age-associated IDD in Ercc1(-/Δ) mice. Collectively, these data provide strong experimental evidence that mitochondrial-derived ROS play a causal role in driving changes linked to aging-related IDD and a potentially important role for radical scavengers in preventing IDD.
10.1002/jor.22320
Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc.
Roberts S,Caterson B,Menage J,Evans E H,Jaffray D C,Eisenstein S M
Spine
STUDY DESIGN:A comprehensive immunohistochemical study of matrix metalloproteinase activity in discs from patients with different disc diseases. OBJECTIVES:To identify individual matrix metalloproteinase enzymes that could contribute to the degeneration of the matrix of the intervertebral disc, to identify the cells that produce matrix metalloproteinases (for example, the endogenous disc cells or invading cells associated with vascularisation), and to determine if "aggrecanase" contributes to degradation of proteoglycans in disc disorders. SUMMARY OF BACKGROUND DATA:Matrix disorganization and loss of substance are the most common findings in degenerate discs, and proteinase enzyme activity is one means of causing these changes. METHODS:Forty-nine discs from 46 patients with degenerative disc disease, posterior anular tears, spondylolisthesis, or disc herniation were studied immunohistochemically to determine the presence of matrix metalloproteinases 1, 2, 3, 7, 8, 9 and 13, tissue metalloproteinases 1 and 2, and proteoglycan degradation products generated by either matrix metalloproteinases or aggrecanase activity. In addition, in situ zymography was used to confirm matrix metalloproteinase activity. RESULTS:The most extensive staining was seen for matrix metalloproteinases 1, 2, 3, and 9, with 91%, 71%, 65%, and 72% of samples having some immunopositivity for the respective antibodies. In contrast, staining for matrix metalloproteinases 7 and 8 was much less (38% for both). Tissue inhibitor of metalloproteinases 1 and 2 were expressed in 34% and 79% of specimens, respectively. Matrix metalloproteinases were found particularly in cell clusters and blood vessels of degenerate discs, with staining correlating positively with macroscopic degenerative grade. For all of the enzymes, there was most staining in the herniation specimens and least in the autopsy samples. The opposite was true of staining for the matrix metalloproteinases inhibitor, tissue inhibitor of metalloproteinases 2, with most found in the autopsy specimens. Enzyme activity was confirmed by in situ zymography and staining for matrix metalloproteinase degradation products of proteoglycans. In addition, there was staining with antibodies demonstrating aggrecanase degradation products. CONCLUSIONS:Matrix metalloproteinase activity is more prevalent in herniated discs than in other disc disorders studied, although matrix metalloproteinases may have been more common earlier in the disease progression. Matrix metalloproteinases can be produced by invading blood vessels and associated cells, as well as by indigenous disc cells. Aggrecanase activity, although present in some samples, was not as obvious as that of matrix metalloproteinases. In addition to altered matrix metalloproteinase production, there appears to be a change in the balance between enzymes and endogenous inhibitors, tissue inhibitors of metalloproteinases. This study highlights specific matrix metalloproteinases that might be most efficient to target in developing therapeutics for minimizing degradation of the extracellular matrix of the disc.
In vivo delivery of MMP3-shRNA and Sox9 lentivirus cocktail enhances matrix synthesis to prevent lumbar disc degeneration.
Zhao Zheng,Li Siyuan,Huang Hui,Fang Jing,Wei Huawei,Xi Yongming
Advances in clinical and experimental medicine : official organ Wroclaw Medical University
BACKGROUND:Intervertebral disc degeneration (IDD) is characterized by increased proteolytic degradation of the extracellular matrix (ECM), leading to a loss of collagen II and proteoglycan in the nucleus pulposus (NP). Although MMP3 has been reported to play a central role in disc degeneration, it is still unknown whether gene therapy targeting MMP3 can inhibit IDD. OBJECTIVES:To investigate whether lentivirus-mediated MMP3 knockdown is capable of attenuating IDD. More importantly, we also explored whether combined gene therapy that simultaneously antagonizes MMP3 and overexpresses Sox9 can synergistically inhibit IDD and induce augmented matrix reconstitution in the degenerative NP. MATERIAL AND METHODS:We performed direct injection of lentiviral vectors LV-MMP3-shRNA and/or LV-Sox9 into rabbit lumbar discs. The animals were scanned using magnetic resonance imaging (MRI) at 8, 12 and 24 weeks after the operation. We also evaluated the gene expression and synthesis of NP matrix components, including collagen II, aggrecan and proteoglycan. RESULTS:The MRI scans showed remarkable needle-puncture-induced progressive IDD in animals injected with PBS or 10^7 viral particles (VP) of the control virus. In contrast, injection of 10^7 VP of LV-MMP3-shRNA or LV-Sox9 substantially inhibited IDD. MMP3 knockdown or Sox9 overexpression stimulated collagen II and aggrecan expression, as well as proteoglycan synthesis. Notably, the injection of a cocktail of LV-MMP3-shRNA and LV-Sox9 (5 × 10^6 VP each) greatly delayed the development of IDD and induced the highest levels of collagen II and proteoglycan production, indicating a synergistic effect in ECM induction. CONCLUSIONS:Our results suggest that gene therapy targeting MMP3 is an efficient way to delay IDD. Combined gene therapy possesses a stronger capacity to induce matrix components in degenerative NP tissue than single-gene delivery.
10.17219/acem/121509
Matrix Degradative Enzymes and Their Inhibitors during Annular Inflammation: Initial Step of Symptomatic Intervertebral Disc Degeneration.
Kim Joo Han,Park Jin Hyun,Moon Hong Joo,Kwon Taek Hyun,Park Youn Kwan
Journal of Korean Neurosurgical Society
OBJECTIVE:Symptomatic disc degeneration develops from inflammatory reactions in the annulus fibrosus (AF). Although inflammatory mediators during annular inflammation have been studied, the roles of matrix metalloproteinases (MMPs) and their inhibitors have not been fully elucidated. In this study, we evaluated the production of MMPs and tissue inhibitors of metalloproteinase (TIMPs) during annular inflammation using an in vitro co-culture system. We also examined the effect of notochordal cells on annular inflammation. METHODS:Human AF (hAF) pellet was co-cultured for 48 hours with phorbol myristate acetate-stimulated macrophage-like THP-1 cells. hAF pellet and conditioned media (CM) from co-cultured cells were assayed for MMPs, TIMPs, and insulin-like growth factor (IGF)-1 levels using real-time reverse-transcriptase polymerase chain reaction and enzyem-linked immunosorbent assay. To evaluate whether notochordal cells affected MMPs or TIMPs production on annular inflammation, hAF co-cultured with notochordal cells from adult New Zealand White rabbits, were assayed. RESULTS:MMP-1, -3, -9; and TIMP-1 levels were significantly increased in CM of hAF co-cultured with macrophage-like cells compared with hAF alone, whereas TIMP-2 and IGF-1 levels were significantly decreased (p<0.05). After macrophage exposure, hAF produced significantly more MMP-1 and -3 and less TIMP-1 and -2. Interleukin-1β stimulation enhanced MMP-1 and -3 levels, and significantly diminished TIMP-2 levels. Co-culturing with rabbit notochordal cells did not significantly influence MMPs and TIMPs production or COL1A2 gene expression. CONCLUSION:Our results indicate that macrophage-like cells evoke annular degeneration through the regulation of major degradative enzymes and their inhibitors, produced by hAF, suggesting that the selective regulation of these enzymes provides future targets for symptomatic disc degeneration therapy.
10.3340/jkns.2014.55.5.237
Aspirin-Mediated Attenuation of Intervertebral Disc Degeneration by Ameliorating Reactive Oxygen Species and .
Oxidative medicine and cellular longevity
Intervertebral disc (IVD) degeneration (IDD) is a major cause of low back pain. The pathogenesis of IDD is associated with the disturbance of reactive oxygen species (ROS) equilibrium, inflammation, and matrix loss. Aspirin is a nonsteroidal anti-inflammatory drug that effectively inhibits inflammation and oxidative stress and has been widely used for the treatment of back pain. Therefore, we hypothesize that aspirin reverses the IDD process via antioxidative and anti-inflammatory effects on the AMPK signaling pathway. , aspirin diminished cellular oxygen free radicals (ROS, nitric oxide (NO)) and inflammatory cytokines (interleukin- (IL-) 1 and IL-6 and tumor necrosis factor alpha (TNF-)) induced by lipopolysaccharides (LPS) in nucleus pulposus cells (NPCs). We found that aspirin preserved the extracellular matrix (ECM) content of collagen type II (COL2) and aggrecan while inhibiting the expression of matrix-degenerating enzymes, including matrix metalloproteinase 3 and 13 (MMP-3 and MMP-13) and A disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS-4, ADAMTS-5). Aspirin significantly promoted the ratios of p-AMPK to AMPK and p-ACC to ACC expression in NPCs. Furthermore, pretreatment with the AMPK inhibitor compound C abrogated the antioxidant effects of aspirin. , an IDD model was established in Sprague-Dawley rats via percutaneous disc puncture with the 20-gauge needle on levels 8-9 and 9-10 of the coccygeal vertebrae. Imaging assessment showed that after aspirin treatment, improvements in disc height index (DHI) ranged from 1.22-fold to 1.54-fold and nucleus pulposus signal strength improved from 1.26-fold to 1.33-fold. Histological analysis showed that aspirin treatment prevented the loss of COL2 and decreased MMP-3 and MMP-13, inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1, and TNF- expression in the IVD tissues. These results suggest that treatment with aspirin could reverse the IDD process via the AMPK signaling pathway, which provides new insights into the potential clinical applications of aspirin, particularly for IDD treatment.
10.1155/2019/7189854
Increased expression of matrix metalloproteinase-10, nerve growth factor and substance P in the painful degenerate intervertebral disc.
Richardson Stephen M,Doyle Paul,Minogue Ben M,Gnanalingham Kanna,Hoyland Judith A
Arthritis research & therapy
INTRODUCTION:Matrix metalloproteinases (MMPs) are known to be involved in the degradation of the nucleus pulposus (NP) during intervertebral disc (IVD) degeneration. This study investigated MMP-10 (stromelysin-2) expression in the NP during IVD degeneration and correlated its expression with pro-inflammatory cytokines and molecules involved in innervation and nociception during degeneration which results in low back pain (LBP). METHODS:Human NP tissue was obtained at postmortem (PM) from patients without a history of back pain and graded as histologically normal or degenerate. Symptomatic degenerate NP samples were also obtained at surgery for LBP. Expression of MMP-10 mRNA and protein was analysed using real-time polymerase chain reaction and immunohistochemistry. Gene expression for pro-inflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha), nerve growth factor (NGF) and the pain-associated neuropeptide substance P were also analysed. Correlations between MMP-10 and IL-1, TNF-alpha and NGF were assessed along with NGF with substance P. RESULTS:MMP-10 mRNA was significantly increased in surgical degenerate NP when compared to PM normal and PM degenerate samples. MMP-10 protein was also significantly higher in degenerate surgical NP samples compared to PM normal. IL-1 and MMP-10 mRNA demonstrated a significant correlation in surgical degenerate samples, while TNF-alpha was not correlated with MMP-10 mRNA. NGF was significantly correlated with both MMP-10 and substance P mRNA in surgical degenerate NP samples. CONCLUSIONS:MMP-10 expression is increased in the symptomatic degenerate IVD, where it may contribute to matrix degradation and initiation of nociception. Importantly, this study suggests differences in the pathways involved in matrix degradation between painful and pain-free IVD degeneration.
10.1186/ar2793
miR‑654‑5p inhibits autophagy by targeting ATG7 via mTOR signaling in intervertebral disc degeneration.
Molecular medicine reports
Intervertebral disc degeneration (IDD) is a common chronic disease characterized by the loss of extracellular matrix (ECM) in the nucleus pulposus (NP). Accumulating evidence has revealed that abnormal expression of microRNAs (miRs) is closely associated with IDD development. The present study aimed to investigate the precise role and possible mechanism underlying the effects of miR‑654‑5p in the pathogenesis of IDD. NP cells were isolated from patients with IDD. Monodansylcadaverine staining was conducted to reveal cell autophagy, while western blotting was performed to detect the expression of ECM‑related proteins in NP cells. Luciferase reporter and RNA immunoprecipitation assays were conducted to identify the binding between RNAs. The results demonstrated that miR‑654‑5p was significantly upregulated in degenerated NP tissues from patients with IDD and high miR‑654‑5p expression was positively associated with disc degeneration grade. Functional assays suggested that miR‑654‑5p facilitated ECM degradation by increasing the expression levels of MMP‑3, MMP‑9 and MMP‑13, as well as decreasing collagen I, collagen II, SOX9 and aggrecan expression by inhibiting autophagy. Furthermore, autophagy‑related gene 7 (ATG7) was verified as a direct downstream target gene of miR‑654‑5p. miR‑654‑5p could bind to the 3' untranslated region of ATG7 to inhibit its mRNA expression and further reduce its translation. Notably, ATG7 knockdown abrogated the effects of the miR‑654‑5p inhibitor on ECM degradation and autophagy regulation. Furthermore, miR‑654‑5p inhibited autophagy in NP cells by increasing the protein expression levels of phosphorylated (p)‑PI3K, p‑AKT and p‑mTOR in an ATG7‑dependent manner. In conclusion, the results of the present study revealed that miR‑654‑5p may enhance ECM degradation via inhibition of autophagy by targeting ATG7 to activate the PI3K/AKT/mTOR signaling pathway. These findings may provide novel insights into the treatment of IDD.
10.3892/mmr.2021.12083
Evaluation of Common Variants in Matrix Metalloproteinase-9 Gene with Lumbar Disc Herniation in Han Chinese Population.
Jing Rong,Liu Yunlei,Guo Peng,Ni Tong,Gao Xiang,Mei Rong,He Xijing,Zhang Jianlin
Genetic testing and molecular biomarkers
OBJECTIVE:Lumbar disc herniation (LDH) is a common and frequent orthopedic disease with strong genetic determinants. The disruption of the intervertebral disc extracellular matrix has been found to play a key role in the development of LDH, suggesting that abnormal matrix metalloproteinases (MMPs) may promote the degradation of the disc matrix. MMP-9, an important member of the MMP family, is a good candidate for the LDH susceptibility gene. The present study aimed to investigate the association of common variants in the MMP-9 gene with the risk, severity, and clinical characteristic variables of LDH. MATERIALS AND METHODS:Fourteen tag single nucleotide polymorphisms (SNPs) entirely covering the region of the MMP-9 gene were analyzed in a sample of 845 patients and 1751 healthy controls. RESULTS:The SNP rs17576 was found to be significantly associated with susceptibility to LDH (OR = 0.77, p = 0.0002), which was also confirmed by haplotype-based analyses (rs79845319-rs17576-rs45437897, global p < 0.001). Our results indicated that the A allele of rs17576 reduced the risk of LDH by ∼23% on average. Furthermore, the G allele of rs17576 was found to correlate with more severe grades of disc degeneration. CONCLUSION:Our results provide additional evidence supporting an important role of the MMP-9 gene in the pathogenesis of LDH.
10.1089/gtmb.2018.0080
Role of miR-155 in the regulation of MMP-16 expression in intervertebral disc degeneration.
Zhang Wei-Lin,Chen Yu-Fei,Meng Hong-Zheng,Du Jun-Jie,Luan Guan-Nan,Wang Hai-Qiang,Yang Mao-Wei,Luo Zhuo-Jing
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
The molecular mechanisms of intervertebral disc degeneration (IDD) remain elusive. We found that miR-155 is down-regulated in degenerative nucleus pulposus (NP), and more severe degeneration is correlated with higher matrix metallopeptidase 16 (MMP-16) expression. MMP-16 also degraded matrix aggrecan. Here, we addressed the in vivo miR-155-mediated pathological impact on IDD using a classic puncture mouse model. Lentiviral upregulated-miR-155 or downregulated-miR-155 was transduced into the discs of C57 mice, which was validated by real-time polymerase chain reaction (real-time PCR) and in situ hybridization. Immunohistochemistry and western blotting revealed that up-regulation of miR-155 resulted in down-regulation of MMP-16 and an increase in aggrecan and collagen type II in mouse NP; whereas, down-regulation of miR-155 resulted in up-regulation of MMP-16 and a decrease in aggrecan in mouse NP. Radiographic and histological analysis showed that the up-regulation of miR-155 attenuated IDD, while down-regulation of miR-155 resulted in the deterioration of IDD. These findings indicate that decreased miR-155 contributed to the up-regulation of MMP-16 in vivo, and MMP-16 further degraded aggrecan and collagen type II, leading to the dehydration and degeneration of discs. Our findings revealed a therapeutic role for miR-155 in IDD. © 2017 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1323-1334, 2017.
10.1002/jor.23313
Prolonged spinal loading induces matrix metalloproteinase-2 activation in intervertebral discs.
Hsieh Adam H,Lotz Jeffrey C
Spine
STUDY DESIGN:An established in vivo mouse model of compression-induced disc degeneration was used to investigate the effects of load on matrix catabolism. OBJECTIVES:To determine whether matrix metalloproteinase-2 expression in discs is modulated by mechanical load and to characterize the regulation of matrix metalloproteinase-2 activity. SUMMARY OF BACKGROUND DATA:We have previously shown that static compression of discs elicits changes in tissue architecture consistent with those seen with degeneration. Evidence in the literature demonstrates the existence of matrix metalloproteinases in both healthy and pathologic discs and suggests that mechanical load may influence matrix metalloproteinase expression and activity. METHODS:Static compression was applied to mouse coccygeal discs in vivo for 1, 4, or 7 days, with adjacent discs serving as sham control. An activity assay was used to measure concentrations of active and total matrix metalloproteinase-2, and changes in matrix metalloproteinase-2 gene expression relative to beta-actin were assessed by reverse transcriptase-polymerase chain reaction. RESULTS:Although no change was seen relative to sham after 1 day of load, the proportion of total matrix metalloproteinase-2 that was active increased after 4 days. This elevation was sustained through 7 days of compression, with no significant differences in total matrix metalloproteinase-2 concentrations among discs throughout the range of time points examined. Semiquantitative reverse transcriptase-polymerase chain reaction demonstrated no significant changes in matrix metalloproteinase-2 gene expression at 1 day or 4 days. CONCLUSIONS:In this model, regulation of matrix metalloproteinase-2 activity occurs primarily through enhanced molecular activation of the proenzyme rather than through elevated gene expression or translation. Our results suggest that matrix metalloproteinase-2 may have a role in load-induced changes in disc architecture.
10.1097/01.BRS.0000083282.82244.F3
PPAR-γ agonist pioglitazone protects against IL-17 induced intervertebral disc inflammation and degeneration via suppression of NF-κB signaling pathway.
Liu Yi,Qu Yuan,Liu Lian,Zhao Hua,Ma Hecheng,Si Meng,Cheng Lei,Nie Lin
International immunopharmacology
Interleukin-17 (IL-17) is the production of T helper type 17 (Th17) cells and has been reported to play a pro-inflammatory role in the immunopathogenesis of intervertebral disc degeneration. Peroxisome proliferator-activated receptor γ (PPAR-γ) activators display anti-inflammatory and anti-degeneration roles in osteoarthritis and rheumatoid arthritis. However, the expression level of PPAR-γ and related regulatory mechanisms in the nucleus pulposus tissues are not clear. Herein we report that PPAR-γ was down-regulated both in the nucleus pulposus tissue of intervertebral disc degeneration patient and in the cultured nucleus pulposus cells stimulated with IL-17. This study was undertaken to investigate the potential therapeutic effect of pioglitazone, as a PPAR-γ ligand, and its underlying molecular mechanism in IL-17-induced human intervertebral disc degeneration model in vitro. Our results indicate that pioglitazone administration suppressed the production of pro-inflammatory cytokines and down-regulated the mRNA expression levels of inflammatory mediators in the cultured human nucleus pulposus cells and tissue. Consistently, pioglitazone decreased the levels of metalloproteinase and maintained the expression of critical matrix components, such as aggrecan and type II collagen. Moreover, the activation of NF-κB signaling in the nucleus pulposus tissue during the intervertebral disc degeneration development was antagonized by pioglitazone administration. In conclusion, our current findings provide scientific evidence for the assessment of pioglitazone as a potential therapeutic approach to treat the intervertebral disc degeneration.
10.1016/j.intimp.2019.04.012
A selective inhibition of c-Fos/activator protein-1 as a potential therapeutic target for intervertebral disc degeneration and associated pain.
Makino Hiroto,Seki Shoji,Yahara Yasuhito,Shiozawa Shunichi,Aikawa Yukihiko,Motomura Hiraku,Nogami Makiko,Watanabe Kenta,Sainoh Takeshi,Ito Hisakatsu,Tsumaki Noriyuki,Kawaguchi Yoshiharu,Yamazaki Mitsuaki,Kimura Tomoatsu
Scientific reports
Intervertebral disc (IVD) degeneration is a major cause of low back pain. The transcription factor c-Fos/Activator Protein-1 (AP-1) controls the expression of inflammatory cytokines and matrix metalloproteinases (MMPs) that contribute to the pathogenesis IVD degeneration. We investigated the effects of inhibition of c-Fos/AP-1 on IVD degeneration and associated pain. A selective inhibitor, T-5224, significantly suppressed the interleukin-1β-induced up-regulation of Mmp-3, Mmp-13 and Adamts-5 transcription in human nucleus pulposus cells and in a mouse explant culture model of IVD degeneration. We used a tail disc percutaneous needle puncture method to further assess the effects of oral administration of T-5224 on IVD degeneration. Analysis of disc height, T2-magnetic resonance imaging (MRI) findings, and histology revealed that IVD degeneration was significantly mitigated by T-5224. Further, oral administration of T-5224 ameliorated pain as indicated by the extended tail-flick latency in response to heat stimulation of rats with needle-puncture-induced IVD degeneration. These findings suggest that the inhibition of c-Fos/AP-1 prevents disc degeneration and its associated pain and that T-5224 may serve as a drug for the prevention of IVD degeneration.
10.1038/s41598-017-17289-y
Mechanical destabilization induced by controlled annular incision of the intervertebral disc dysregulates metalloproteinase expression and induces disc degeneration.
Melrose James,Shu Cindy,Young Cara,Ho Ronald,Smith Margaret M,Young Allan A,Smith Susan S,Gooden Ben,Dart Andrew,Podadera Juan,Appleyard Richard C,Little Christopher B
Spine
STUDY DESIGN:An investigation of mechanical destabilization of the lumbar ovine intervertebral disc (IVD) inducing IVD degeneration (IVDD) as determined by multiparameter outcome measures (magnetic resonance imaging [MRI], IVD composition, biomechanical testing, gene profiling, immunohistochemistry, and immunoblotting). OBJECTIVE:To assess the effect of IVD mechanical destabilization on matrix protein and metalloproteinase gene expression to investigate the pathophysiological mechanisms of lumbar IVDD. SUMMARY OF BACKGROUND DATA:Several earlier studies have used annular transection to induce IVDD in sheep, but none have optimized or validated the most appropriate lesion size. METHODS:The annulus fibrosus (AF) incision inducing maximal change in IVD biomechanics was applied to L1-L2, L3-L4, and L5-L6 discs in vivo to compare with a sham procedure at 3 months post operation. IVDs were evaluated by MRI, biomechanics, histopathology, proteoglycan and collagen content, gene expression, and aggrecan proteolysis by Western blotting. RESULTS:Significant changes were observed in lesion (6 × 20 mm(2)) compared with sham IVDs at 3 months post operation: reduced disc height on MRI; increased neutral zone in biomechanical testing; depleted proteoglycan and collagen content in the nucleus pulposus (NP) and lesion half of the AF but not in the contralateral AF; increased messenger RNA for collagen I and II, aggrecan, versican, perlecan, matrix metalloproteinase (MMP)-1 & 13, and ADAMTS-5, in the lesion-site AF and NP but not in the contralateral AF. ADAMTS-4 messenger RNA was increased in the lesion-site AF but decreased in the NP. Despite an upregulation in MMPs, there was no change in MMP- or ADAMTS-generated aggrecan neoepitopes in any region of the IVD in lesion or sham discs. CONCLUSION:Lumbar IVDD was reproducibly induced with a 6 × 20 mm(2) annular lesion, with focal dysregulation of MMP gene expression, cell cloning in the inner AF, loss of NP aggrecan, and disc height. Loss of aggrecan from the NP was not attributable to increased proteolysis in the interglobular domain by MMPs or ADAMTS.
10.1097/BRS.0b013e31820cd8d5
Influences of circulatory factors on intervertebral disc aging phenotype.
Lei Changbin,Colangelo Debora,Patil Prashanti,Li Vivian,Ngo Kevin,Wang Dong,Dong Qing,Yousefzadeh Matthew J,Lin Hongsheng,Lee Joon,Kang James,Sowa Gwendolyn,Wyss-Coray Tony,Niedernhofer Laura J,Robbins Paul D,Huffman Derek M,Vo Nam
Aging
Whether disc aging is influenced by factors beyond its local environment is an important unresolved question. Here we performed heterochronic parabiosis in mice to study the effects of circulating factors in young and old blood on age-associated intervertebral disc degeneration. Compared to young isochronic pairs (Y-Y), young mice paired with old mice (Y-O) showed significant increases in levels of disc MMP-13 and ADAMTS4, aggrecan fragmentation, and histologic tissue degeneration, but negligible changes in cellular senescence markers (p16, p21). Compared to old isochronic pairs (O-O), old mice paired with young mice (O-Y) exhibited a significant decrease in expression of cellular senescence markers (p16, p21, p53), but only marginal decreases in the levels of disc MMP-13 and ADAMTS4, aggrecan fragmentation, and histologic degeneration. Thus, exposing old mice to young blood circulation greatly suppressed disc cellular senescence, but only slightly decreased disc matrix imbalance and degeneration. Conversely, exposing young mice to old blood accelerated their disc matrix imbalance and tissue degeneration, with little effects on disc cellular senescence. Thus, non-cell autonomous effects of circulating factors on disc cellular senescence and matrix homeostasis are complex and suggest that disc matrix homeostasis is modulated by systemic factors and not solely through local disc cellular senescence.
10.18632/aging.103421
Dysregulated miR-133a Mediates Loss of Type II Collagen by Directly Targeting Matrix Metalloproteinase 9 (MMP9) in Human Intervertebral Disc Degeneration.
Xu Yun-Qiang,Zhang Zhen-Hui,Zheng Yong-Fa,Feng Shi-Qing
Spine
STUDY DESIGN:A microRNA (miRNA) study using Solexa sequencing. OBJECTIVE:The purpose of this study was to identify intervertebral disc degeneration (IDD)-specific miRNA expression profile, and to validate its biological function. SUMMARY OF BACKGROUND DATA:Accumulating evidence indicates that miRNAs play a critical role in IDD, but the role of specific miRNAs involved in this entity remains unclear. METHODS:MiRNA expression profile was determined in nucleus pulposus (NP) tissues from patients with IDD and controls, employing Solexa sequencing and quantitative real-time PCR (qRT-PCR). Biological functions of differential expression miRNAs were further investigated. Luciferase reporter assays and western blotting were performed to determine miRNA targets. RESULTS:We identified 31 miRNAs that were differentially expressed (22 upregulated and nine downregulated) in patients compared with controls. After qRT-PCR confirmation, miR-133a was significantly down-regulated in degenerative NP tissues. Moreover, its level was inversely correlated with grade of disc degeneration. Through gain- and loss-of-function studies, miR-133a was demonstrated to significantly promote type II collagen expression in NP cells. MMP9 was identified as a target of miR-133a. Knockdown of MMP9 induced effects on NP cells similar to those induced by miR-133a. Expression of MMP9 was inversely correlated with miR-133a expression in degenerative NP tissues. CONCLUSION:These results suggest that the downregulation of miR-133a induces type II collagen loss by directly targeting MMP9. Our findings also highlight miR-133a as a novel hopeful therapeutic target for IDD. LEVEL OF EVIDENCE:3.
10.1097/BRS.0000000000001375
Matrix metalloproteinase-3 production by human degenerated intervertebral disc.
Nemoto O,Yamagishi M,Yamada H,Kikuchi T,Takaishi H
Journal of spinal disorders
To assess its possible role in the pathophysiology of intervertebral disc degeneration, we investigated the production of matrix metalloproteinase-3 (MMP-3) using human intervertebral disc explant culture. Five normal and 10 degenerated disc specimens were used. The levels of MMP-3 released in the medium were measured with use of an enzyme immunoassay. The results showed that the level of MMP-3 in the degenerated group (0.57 microg/ml/mg wet weight; n = 10) was significantly higher than that of the control group (0.29 microg/ml/mg wet weight; n = 5) (p < 0.05). Immunostaining of MMP-3 revealed that the ratio of positive staining cells in the degenerated group was greater than that of the control group. These observations suggest that MMP-3 produced by human intervertebral disc may be involved in the intervertebral disc degeneration, particularly in the initiation of matrix degradation of intervertebral disc.
LIM mineralization protein-1 suppresses TNF-α induced intervertebral disc degeneration by maintaining nucleus pulposus extracellular matrix production and inhibiting matrix metalloproteinases expression.
Liu Hui,Pan Hehai,Yang Hao,Wang Jianru,Zhang Kuibo,Li Xiang,Wang Hua,Ding Wenbin,Li Bingxue,Zheng Zhaomin
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Imbalanced metabolism of Nucleus pulposus (NP) extracellular matrix (ECM) is closely correlated to Intervertebral Disc Degenerative Disease. LIM mineralization protein-1 (LMP-1) has been proven to induce sulfated glycosaminoglycan (sGAG) production in NP and have an anti-inflammatory effect in pre-osteoclast. However, whether it has any effect on the NP ECM production and degradation under inflammatory stimulation has not been studied. In the current study, a TNF-α induced cell model was established in vitro. Lentivirus encoding LMP-1 (LV-LMP-1) and short heparin LMP-1 (LV-shLMP-1) were constructed to overexpress and knockdown LMP-1 expression in NP cells. LMP-1 mRNA level was regulated in a dose-dependent manner after transfection. LV-LMP-1 increased whereas LV-shLMP-1 decreased collagen II, aggrecan, versican expression, and sGAG production. LV-LMP-1 abolished while LV-shLMP-1 aggravated TNF-α mediated down-regulation of the above matrix genes via ERK1/2 activation. Moreover, LV-LMP-1 abrogated TNF-α induced MMP-3 and MMP-13 expression via inhibiting p65 translocation and MMP-3 and MMP-13 promoter activity. These results indicated that LMP-1 had an ECM production maintenance effect under inflammatory stimulation. This effect was via up-regulation of matrix genes expression at least partially through ERK1/2 activation, and down-regulation of MMPs expression through NF-κB inhibition.
10.1002/jor.22732
Loss of tenomodulin expression is a risk factor for age-related intervertebral disc degeneration.
Aging cell
The intervertebral disc (IVD) degeneration is thought to be closely related to ingrowth of new blood vessels. However, the impact of anti-angiogenic factors in the maintenance of IVD avascularity remains unknown. Tenomodulin (Tnmd) is a tendon/ligament-specific marker and anti-angiogenic factor with abundant expression in the IVD. It is still unclear whether Tnmd contributes to the maintenance of IVD homeostasis, acting to inhibit vascular ingrowth into this normally avascular tissue. Herein, we investigated whether IVD degeneration could be induced spontaneously by the absence of Tnmd. Our results showed that Tnmd was expressed in an age-dependent manner primarily in the outer annulus fibrous (OAF) and it was downregulated at 6 months of age corresponding to the early IVD degeneration stage in mice. Tnmd knockout (Tnmd ) mice exhibited more rapid progression of age-related IVD degeneration. These signs include smaller collagen fibril diameter, markedly lower compressive stiffness, reduced multiple IVD- and tendon/ligament-related gene expression, induced angiogenesis, and macrophage infiltration in OAF, as well as more hypertrophic-like chondrocytes in the nucleus pulposus. In addition, Tnmd and chondromodulin I (Chm1, the only homologous gene to Tnmd) double knockout (Tnmd Chm1 ) mice displayed not only accelerated IVD degeneration, but also ectopic bone formation of IVD. Lastly, the absence of Tnmd in OAF-derived cells promoted p65 and matrix metalloproteinases upregulation, and increased migratory capacity of human umbilical vein endothelial cells. In sum, our data provide clear evidences that Tnmd acts as an angiogenic inhibitor in the IVD homeostasis and protects against age-related IVD degeneration. Targeting Tnmd may represent a novel therapeutic strategy for attenuating age-related IVD degeneration.
10.1111/acel.13091
Immunohistochemical study of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 human intervertebral discs.
Kanemoto M,Hukuda S,Komiya Y,Katsuura A,Nishioka J
Spine
STUDY DESIGN:Immunohistologic staining of human intervertebral discs collected at the time of surgery (100 intervertebral discs from 80 patients) and 10 discs collected from 7 cadavers within 12 hours of death was performed using antimatrix metalloproteinase-3 monoclonal antibody and antitissue inhibitor of metalloproteinase-1 monoclonal antibody. OBJECTIVES:To examine the relationship between matrix destruction and staining for matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 in intervertebral disc degeneration. SUMMARY OF BACKGROUND DATA:Matrix metalloproteinase-3, which decomposes aggregating proteoglycans, has attracted research attention as a substance contributing to matrix destruction in the articular cartilage and intervertebral disc. However, except for a few in vitro studies, the relationship between matrix destruction of the intervertebral disc and matrix metalloproteinase-3 has been little studied. METHODS:Immunohistologic staining was performed to examine the relationship between matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 in the intervertebral disc, and the relationship of these two agents to magnetic resonance imaging, radiographic, and surgical findings. RESULTS:Those cases testing positive for matrix metalloproteinase-3 and negative for tissue inhibitor of metalloproteinase-1 accounted for most of the surgical specimens. The matrix metalloproteinase-3-positive cell ratio was significantly correlated with the magnetic resonance imaging grade of intervertebral disc degeneration, and the matrix metalloproteinase-3-positive cell ratio observed in prolapsed lumbar intervertebral discs was significantly higher than that in nonprolapsed discs. In cervical intervertebral discs, the matrix metalloproteinase-3-positive cell ratio and staining of cartilaginous endplate were correlated with the size of osteophyte formation. CONCLUSIONS:These findings suggested that intervertebral disc degeneration is caused by disturbance in the equilibrium of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1, and that matrix metalloproteinase-3 contributes to degeneration of the cartilaginous endplate.
Toward a biochemical understanding of human intervertebral disc degeneration and herniation. Contributions of nitric oxide, interleukins, prostaglandin E2, and matrix metalloproteinases.
Kang J D,Stefanovic-Racic M,McIntyre L A,Georgescu H I,Evans C H
Spine
STUDY DESIGN:Normal and herniated human intervertebral disc specimens were cultured to study the effects of interleukin-1 beta on the production of nitric oxide, interleukin-6, prostaglandin E2, and matrix metalloproteinases. The effects of endogenously produced nitric oxide on the synthesis of other mediators also were studied. OBJECTIVES:To test the hypothesis that the cells of the intervertebral disc are metabolically active and are capable of responding to biochemical stimuli such as interleukin-1 beta in a manner that could engender degenerative changes. As part of this study, the authors also investigated some of the possible autocrine regulatory mechanisms that may operate during the biochemical responses of disc cells. SUMMARY OF BACKGROUND DATA:The authors previously showed, for the first time, that herniated cervical and lumbar disc specimens spontaneously produce increased amounts of nitric oxide, interleukin-6, prostaglandin E2, and certain matrix metalloproteinases. These results suggest that these biochemical agents are in some manner involved with degenerative processes in the intervertebral disc. This novel hypothesis merits further evaluation; the current communication reports the results of experiments designed to do so. METHODS:Fourteen normal, nondegenerated discs (control group) were obtained from seven patients undergoing anterior spinal surgery for trauma or lumbar scoliosis. Thirty-six herniated discs (18 lumbar and 18 cervical) were obtained from 30 patients undergoing surgery for persistent radiculopathy. The specimens were placed into tissue culture and incubated for 72 hours in the presence or absence of interleukin-1 beta and NG-monomethyl-L-arginine, and inhibitor of nitric oxide synthases, and the media were subsequently collected for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 were performed. RESULTS:Normal, control disc specimens significantly increased their production of matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 in response to interleukin-1 beta. Herniated lumbar and cervical discs, which were spontaneously releasing increased levels of these biochemical agents, further increased their production of nitric oxide, interleukin-6, and prostaglandin E2 in response to interleukin-1 beta. Blocking the biosynthesis of nitric oxide in interleukin-1 beta-stimulated disc cells provoked a large increase in the production of interleukin-6. CONCLUSIONS:Cells of the intervertebral discs are biologically responsive and increase their production of matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 when stimulated by interleukin-1 beta. The effect is more dramatic in normal, nondegenerated discs where spontaneous synthesis of these mediators is low. Nevertheless, cells of the herniated degenerated discs where spontaneous production was high were still capable of further increasing their synthesis of several of these biochemical agents in response to interleukin-1 beta. Endogenously produced nitric oxide appears to have a strong inhibitory effect on the production of interleukin-6, which suggests that autocrine mechanisms play an important role in the regulation of disc cell metabolism.
Effect of Tuina along "bladder meridian" alleviating intervertebral disc degeneration by regulating the transforming growth factor-β1/Smad signaling pathway in a rabbit model.
Journal of traditional Chinese medicine = Chung i tsa chih ying wen pan
OBJECTIVE:The aim of this study was to investigate the protective effects of Tuina (a traditional Chinese massage therapy) on intervertebral disc (IVD) degeneration and the regulatory mechanisms of the transforming growth factor-β1 (TGF-β1)/small mothers against decapentaplegic (Smad) signaling pathway. METHODS:Thirty New Zealand white rabbits were randomized into five groups: the control group, model group, model + Tuina group (Tuina group), model + TGF-β1 group (TGF-β1 group), and model + TGF-β1 inhibitor SB431542 group (SB431542 group). The model was established by posterolateral annulus fibrosus puncturing (AFP). Recombinant TGF-β1 and inhibitor SB431542 was injected into the TGF-β1 group and SB431542 group with a microsyringe, respectively. The rabbits in the Tuina group received Tuina treatment along the bladder meridian for 4 weeks. Magnetic resonance imaging (MRI) was performed on rabbits before AFP and after 4 weeks of intervention. Lumbar IVDs (L2-L3 to L4-L5) were harvested after intervention. Histopathological changes in the IVDs were measured by hematoxylin and eosin (HE) staining. Type I collagen was analyzed by immunohistochemistry detection. The expression level of matrix metalloproteinase-3 (MMP3) was determined by enzyme-linked immunosorbent assay. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated nick end labeling and Western blotting. Real-time polymerase chain reaction and Western blotting were used to analyze the expression of TGF-β1 and Smad2/3/4 and a disintegrin and metalloproteinase with thrombospondin motifs 5. RESULTS:Posterolateral AFP induced IVD degeneration in rabbits with histopathological damage and noticeable changes in MRI images. Tuina alleviated histo-pathological changes and reversed the expression of extracellular matrix degeneration-related molecules and apoptosis-related proteins. Furthermore, AFP induced the activation of TGF-β1 and Smad2/3/4, whereas Tuina therapy markedly reduced the protein expression of Smad2/3 and the gene expression of TGF-β1 and Smad2/3/4. Additionally, the TGF-β1/Smad signaling pathway was activated in the TGF-β1 group, while the TGF-β1/Smad signaling pathway was inhibited in the SB431542 group. CONCLUSION:Posterolateral AFP induced disc degeneration as determined by MRI assessment and histological analysis. Tuina alleviated disc degeneration, possibly by inhibiting the fibrotic response mediated by the TGF-β1/Smad pathway, thus alleviating extracellular matrix degeneration and reducing cell apoptosis.
10.19852/j.cnki.jtcm.2023.05.005
TNF-α induces up-regulation of MicroRNA-27a via the P38 signalling pathway, which inhibits intervertebral disc degeneration by targeting FSTL1.
Journal of cellular and molecular medicine
The mechanism of intervertebral disc degeneration is still unclear, and there are no effective therapeutic strategies for treating this condition. miRNAs are naturally occurring macromolecules in the human body and have many biological functions. Therefore, we hope to elucidate whether miRNAs are associated with intervertebral disc degeneration and the underlying mechanisms involved. In our study, differentially expressed miRNAs were predicted by the GEO database and then confirmed by qPCR and in situ hybridization. Apoptosis of nucleus pulposus cells was detected by flow cytometry and Bcl2, Bax and caspase 3. Deposition of extracellular matrix was assessed by Alcian blue staining, and the expression of COX2 and MMP13 was detected by immunofluorescence, Western blot and qPCR. Moreover, qPCR was used to detect the expression of miR27a and its precursors. The results showed that miR27a was rarely expressed in healthy intervertebral discs but showed increased expression in degenerated intervertebral discs. Ectopic miR27a expression inhibited apoptosis, suppressed the inflammatory response and attenuated the catabolism of the extracellular matrix by targeting FSTL1. Furthermore, it seems that the expression of miR27a was up-regulated by TNF-α via the P38 signalling pathway. So we conclude that TNF-α and FSTL1 engage in a positive feedback loop to promote intervertebral disc degeneration. At the same time, miR27a is up-regulated by TNF-α via the P38 signalling pathway, which ameliorates inflammation, apoptosis and matrix degradation by targeting FSTL1. Thus, this negative feedback mechanism might contribute to the maintenance of a low degeneration load and would be beneficial to maintain a persistent chronic disc degeneration.
10.1111/jcmm.16745
Bone morphogenetic protein-2 provokes interleukin-18-induced human intervertebral disc degeneration.
Ye S,Ju B,Wang H,Lee K-B
Bone & joint research
OBJECTIVES:Interleukin 18 (IL-18) is a regulatory cytokine that degrades the disc matrix. Bone morphogenetic protein-2 (BMP-2) stimulates synthesis of the disc extracellular matrix. However, the combined effects of BMP-2 and IL-18 on human intervertebral disc degeneration have not previously been reported. The aim of this study was to investigate the effects of the anabolic cytokine BMP-2 and the catabolic cytokine IL-18 on human nucleus pulposus (NP) and annulus fibrosus (AF) cells and, therefore, to identify potential therapeutic and clinical benefits of recombinant human (rh)BMP-2 in intervertebral disc degeneration. METHODS:Levels of IL-18 were measured in the blood of patients with intervertebral disc degenerative disease and in control patients. Human NP and AF cells were cultured in a NP cell medium and treated with IL-18 or IL-18 plus BMP-2. mRNA levels of target genes were measured by real-time polymerase chain reaction, and protein levels of aggrecan, type II collagen, SOX6, and matrix metalloproteinase 13 (MMP13) were assessed by western blot analysis. RESULTS:The serum level of patients (IL-18) increased significantly with the grade of IVD degeneration. There was a dramatic alteration in IL-18 level between the advanced degeneration (Grade III to V) group and the normal group (p = 0.008) Furthermore, IL-18 induced upregulation of the catabolic regulator MMP13 and downregulation of the anabolic regulators aggrecan, type II collagen, and SOX6 at 24 hours, contributing to degradation of disc matrix enzymes. However, BMP-2 antagonised the IL-18 induced upregulation of aggrecan, type II collagen, and SOX6, resulting in reversal of IL-18 mediated disc degeneration. CONCLUSIONS:BMP-2 is anti-catabolic in human NP and AF cells, and its effects are partially mediated through provocation of the catabolic effect of IL-18. These findings indicate that BMP-2 may be a unique therapeutic option for prevention and reversal of disc degeneration.Cite this article: S. Ye, B. Ju, H. Wang, K-B. Lee. Bone morphogenetic protein-2 provokes interleukin-18-induced human intervertebral disc degeneration. Bone Joint Res 2016;5:412-418. DOI: 10.1302/2046-3758.59.BJR-2016-0032.R1.
10.1302/2046-3758.59.BJR-2016-0032.R1
Molecular Biological Effects of Weightlessness and Hypergravity on Intervertebral Disc Degeneration.
Wu Di,Zheng Chao,Wu Ji,Huang Rongrong,Chen Xuanyu,Zhang Tong,Zhang Lili
Aerospace medicine and human performance
INTRODUCTION:The rate of intervertebral disc degeneration (IVDD) is influenced by environmental factors. Extracellular matrix (ECM) destruction and apoptosis of intervertebral disc cells are major characteristics of IVDD. ECM degradation is closely linked to up-regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMP). This study aimed to elucidate the molecular biological changes during IVDD under conditions of weightlessness and hypergravity. METHODS:A total of 120 rabbits were divided randomly into four groups: control group, weightlessness group, hypergravity group, and mixed (hypergravity + weightlessness) group. Tail-suspension was used to simulate a weightless environment, and an animal centrifuge (+7 Gz three times for 60 s) was used to mimic hypergravity conditions. After exposure to the above conditions for 30, 60, and 90 d, respectively, 10 rabbits were selected from each group for immunohistochemical determination of MMP-1, MMP-3, and TIMP-1 expression. TUNEL staining was also carried out to detect apoptotic cells in each group at each time point. RESULTS:MMP-1, MMP-3, and TIMP-1 were rarely expressed in the control group, but were positively expressed in the other three groups. The strongest expression was in the mixed group at every time point, followed by the hypergravity group, and then the weightlessness group. Cell apoptosis index followed a similar trend to MMPs and TIMP-1 expression. DISCUSSION:The results suggested that weightlessness and hypergravity may both aggravate IVDD over time, with hypergravity having a particularly marked effect.Wu D, Zheng C, Wu J, Huang R, Chen X, Zhang T, Zhang L. Molecular biological effects of weightlessness and hypergravity on intervertebral disc degeneration. Aerosp Med Hum Perform. 2017; 88(12):1123-1128.
10.3357/AMHP.4872.2017
microRNA-665 promotes the proliferation and matrix degradation of nucleus pulposus through targeting GDF5 in intervertebral disc degeneration.
Tan Hongyu,Zhao Liang,Song Ruipeng,Liu Yilin,Wang Limin
Journal of cellular biochemistry
Growing evidences suggested that microRNAs (miRNAs) played important roles in the development of intervertebral disc degeneration (IDD). However, the expression level and function of miR-665 in IDD remain unknown. In this study, we showed that the expression level of miR-665 was upregulated in degenerative human NP samples. In addition, miR-665 expression level gradually increased with the exacerbation of disc degeneration grade. Moreover, miR-665 expression level was positively associated with the Pfirrmann grade. Ectopic expression of miR-665 promoted NP cell growth. Furthermore, miR-665 overexpression decreased aggrecan and Col II expression and ectopic expression of miR-665 increased MMP-3 and MMP-13 expression in NP cell. We identified growth differentiation factor 5 (GDF5) was a direct target gene of miR-665 in NP cell and enforced expression of miR-665 decreased GDF5 expression. Elevated expression of miR-665 enhanced NP cell proliferation and decreased aggrecan and Col II expression. In addition, ectopic expression of miR-665 increased MMP-3 and MMP-13 expression through inhibiting GDF5 expression in NP cells. These results suggested that dysregulated miR-665 expression might act an important role in the development of IDD.
10.1002/jcb.26888
Microfluidic Electroceuticals Platform for Therapeutic Strategies of Intervertebral Disc Degeneration: Effects of Electrical Stimulation on Human Nucleus Pulposus Cells under Inflammatory Conditions.
International journal of molecular sciences
The degeneration of an intervertebral disc (IVD) is a major cause of lower back pain. IVD degeneration is characterized by the abnormal expression of inflammatory cytokines and matrix degradation enzymes secreted by IVD cells. In addition, macrophage-mediated inflammation is strongly associated with IVD degeneration. However, the precise pathomechanisms of macrophage-mediated inflammation in IVD are still unknown. In this study, we developed a microfluidic platform integrated with an electrical stimulation (ES) array to investigate macrophage-mediated inflammation in human nucleus pulposus (NP). This platform provides multiple cocultures of different cell types with ES. We observed macrophage-mediated inflammation and considerable migration properties via upregulated expression of interleukin (IL)-6 (p < 0.001), IL-8 (p < 0.05), matrix metalloproteinase (MMP)-1 (p < 0.05), and MMP-3 (p < 0.05) in human NP cells cocultured with macrophages. We also confirmed the inhibitory effects of ES at 10 μA due to the production of IL-6 (p < 0.05) and IL-8 (p < 0.01) under these conditions. Our findings indicate that ES positively affects degenerative inflammation in diverse diseases. Accordingly, the microfluidic electroceutical platform can serve as a degenerative IVD inflammation in vitro model and provide a therapeutic strategy for electroceuticals.
10.3390/ijms231710122
MiR-874-3p plays a protective role in intervertebral disc degeneration by suppressing MMP2 and MMP3.
Song Qingxin,Zhang Fan,Wang Kun,Chen Zhi,Li Quan,Liu Zude,Shen Hongxing
European journal of pharmacology
Intervertebral disc degeneration (IDD) is a spinal degenerative disease and one of the most important causes of musculoskeletal disability. Matrix metalloproteinase (MMP)-mediated extracellular matrix degradation is the core process of IDD. The regulators of MMPs in the intervertebral disc are still not fully known. In this study, using quantitative reverse transcription PCR, luciferase reporter assay, Western blotting, immunofluorescence, flow cytometry, and Cell Counting Kit-8 assay, we found that the miR-874-3p expression level was significantly decreased in IDD patients. MiR-874-3p could target and repress MMP2 and MMP3 expression in nucleus pulposus cells. These results could improve the understanding of IDD and provide a possible diagnostic marker and treatment candidate for IDD. The miR-874-3p/MMP2/MMP3 axis might also provide direction for future cancer and inflammation investigations.
10.1016/j.ejphar.2021.173891
microRNA-365 attenuated intervertebral disc degeneration through modulating nucleus pulposus cell apoptosis and extracellular matrix degradation by targeting EFNA3.
Journal of cellular and molecular medicine
This present study is aimed to investigate the role of microRNA-365 (miR-365) in the development of intervertebral disc degeneration (IDD). Nucleus pulposus (NP) cells were transfected by miR-365 mimic and miR-365 inhibitor, respectively. Concomitantly, the transfection efficiency and the expression level of miRNA were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Meanwhile, NP cells apoptosis was measured through propidium iodide (PI)-AnnexinV-fluorescein isothiocyanate (FITC) apoptosis detection kit. Subsequently, immunofluorescence (IF) staining was performed to assess the expression of collagen II, aggrecan and matrix metalloproteinase 13 (MMP-13). In addition, bioinformatic prediction and Luciferase reporter assay were used to reveal the target gene of miR-365. Finally, we isolated the primary NP cells from rats and injected NP-miR-365 in rat IDD models. The results showed that overexpression of miR-365 could effectively inhibit NP cells apoptosis and MMP-13 expression and upregulate the expression of collagen II and aggrecan. Conversely, suppression of miR-365 enhanced NP cell apoptosis and elevated MMP-13 expression, but decreased the expression of collagen II and aggrecan. Moreover, the further data demonstrated that miR-365 mediated NP cell degradation through targeting ephrin-A3 (EFNA3). In addition, the cells apoptosis and catabolic markers were increased in NP cells when EFNA3 upregulated. More importantly, the vivo data supported that miR-365-NP cells injection ameliorated IDD in rats models. miR-365 could alleviate the development of IDD by regulating NP cell apoptosis and ECM degradation, which is likely mediated by targeting EFNA3. Therefore, miR-365 may be a promising therapeutic avenue for treatment IDD through EFNA3.
10.1111/jcmm.18054
The association of degeneration of the intervertebral disc with 5a/6a polymorphism in the promoter of the human matrix metalloproteinase-3 gene.
Takahashi M,Haro H,Wakabayashi Y,Kawa-uchi T,Komori H,Shinomiya K
The Journal of bone and joint surgery. British volume
It has been suggested that matrix metalloproteinase-3 (MMP-3, stromelysin-1) has an important role in the degeneration of intervertebral discs (IVDs). A human MMP-3 promoter 5A/6A polymorphism was reported to be involved in the regulation of MMP-3 gene expression. We suggest that IVD degeneration is associated with 5A/6A polymorphism. We studied 54 young and 49 elderly Japanese subjects. Degeneration of the lumbar discs was graded using MRI in the younger group and by radiography in the elderly. 5A/6A polymorphism was determined by polymerase-chain reaction-based assays. We found that the 5A5A and 5A6A genotype in the elderly was associated with a significantly larger number of degenerative IVDs than the 6A6A (p < 0.05), but there was no significant difference in the young. In the elderly, the IVD degenerative scores were also distributed more highly in the 5A5A and 5A6A genotypes (p = 0.0029). Our findings indicate that the 5A allele is a possible risk factor for the acceleration of degenerative changes in the lumbar disc in the elderly.
Downregulation of miR-27b is Involved in Loss of Type II Collagen by Directly Targeting Matrix Metalloproteinase 13 (MMP13) in Human Intervertebral Disc Degeneration.
Li Hao-ran,Cui Qing,Dong Zhan-yin,Zhang Jian-hua,Li Hai-qing,Zhao Ling
Spine
STUDY DESIGN:A microRNA (miRNA) study. OBJECTIVE:The purpose of this study was to identify intervertebral disc degeneration (IDD)-specific miRNAs, followed by functional validation of results. SUMMARY OF BACKGROUND DATA:IDD is the major contributor to back radicular pain, and the molecular mechanisms underlying this disease are not completely understood. Accumulating evidence suggests that miRNAs play an important role in IDD, but the role of specific miRNAs involved in this disease remains elusive. METHODS:An initial screening of nucleus pulposus (NP) tissues, miRNA expression by miRNA microarray, was performed using samples from 10 patients with degenerative disc disease and 10 patients with lumbar fracture (as controls). Subsequently, differential expression was validated using quantitative reverse transcriptase PCR (qRT-PCR). The level of differentially expressed miRNAs in degenerative NP tissues was investigated, and then functional analysis of the miRNAs in regulating collagen II expression was carried out. Western blotting and luciferase reporter assays were also used to detect the target gene. RESULTS:We identified 23 miRNAs that were differentially expressed (16 upregulated and 7 downregulated) in patients compared with controls. After qRT-PCR confirmation, miR-27b was significantly downregulated in degenerative NP tissues when compared with controls. Moreover, its level was correlated with grade of disc degeneration. Overexpression of miR-27b promoted type II collagen expression in NP cells. Bioinformatics target prediction identified matrix metalloproteinase 13 (MMP13) as a putative target of miR-27b. Futhermore, luciferase reporter assays demonstrated that miR-27b directly targets MMP13 and affects the protein expression of MMP13 in NP cells. Expression of MMP13 negatively correlated with miR-27b expression in degenerative NP tissues. CONCLUSION:The downregulation of miR-27b induces type II collagen loss by directly targeting MMP13, leading to the development of IDD. Our study also underscores the potential of miR-27b as a novel therapeutic target in human IDD. LEVEL OF EVIDENCE:3.
10.1097/BRS.0000000000001139
Glycosaminoglycan synthesis in the nucleus pulposus: Dysregulation and the pathogenesis of disc degeneration.
Matrix biology : journal of the International Society for Matrix Biology
Few human tissues have functions as closely linked to the composition of their extracellular matrices as the intervertebral disc. In fact, the hallmark of intervertebral disc degeneration, commonly accompanying low back and neck pain, is the progressive loss of extracellular matrix molecules - specifically the GAG-substituted proteoglycans. While this loss is often associated with increased extracellular catabolism via metalloproteinases and pro-inflammatory cytokines, there is strong evidence that disc degeneration is related to dysregulation of the enzymes involved in GAG biosynthesis. In this review, we discuss those environmental factors, unique to the disc, that control expression and function of XT-1, GlcAT-I, and ChSy/ChPF in the healthy and degenerative state. Additionally, we address the pathophysiology of aberrant GAG biosynthesis and highlight therapeutic strategies designed to augment the loss of extracellular matrix molecules that afflict the degenerative state.
10.1016/j.matbio.2018.02.025
Enhancement of intervertebral disc cell senescence by WNT/β-catenin signaling-induced matrix metalloproteinase expression.
Hiyama Akihiko,Sakai Daisuke,Risbud Makarand V,Tanaka Masahiro,Arai Fumiyuki,Abe Koichiro,Mochida Joji
Arthritis and rheumatism
OBJECTIVE:To determine whether intervertebral disc (IVD) cells express β-catenin and to assess the role of the WNT/β-catenin signaling pathway in cellular senescence and aggrecan synthesis. METHODS:The expression of β-catenin messenger RNA (mRNA) and protein in rat IVD cells was assessed by using several real-time reverse transcription-polymerase chain reaction, Western blot, immunohistochemical, and immunofluorescence analyses. The effect of WNT/β-catenin on nucleus pulposus (NP) cells was examined by transfection experiments, an MTT assay, senescence-associated β-galactosidase staining, a cell cycle analysis, and a transforming growth factor (TGFβ)/bone morphogenetic protein (BMP) pathway-focused microarray analysis. RESULTS:We found that β-catenin mRNA and protein were expressed in discs in vivo and that rat NP cells exhibited increased β-catenin mRNA and protein upon stimulation with lithium chloride, a known activator of WNT signaling. LiCl treatment inhibited the proliferation of NP cells in a time- and dose-dependent manner. In addition, there was an increased level of cellular senescence in LiCl-treated cells. Long-term treatment with LiCl induced cell cycle arrest and promoted subsequent apoptosis in NP cells. Activation of WNT/β-catenin signaling also regulated the expression of aggrecan. We also demonstrated that WNT/β-catenin signaling induced the expression of matrix metalloproteinases (MMPs) and TGFβ in NP cells. CONCLUSION:The activation of WNT/β-catenin signaling promotes cellular senescence and may modulate MMP and TGFβ signaling in NP cells. We hypothesize that the activation of WNT/β-catenin signaling may lead to an increased breakdown of the matrix, thereby promoting IVD degeneration.
10.1002/art.27599
Loss of TIMP3 expression induces inflammation, matrix degradation, and vascular ingrowth in nucleus pulposus: A new mechanism of intervertebral disc degeneration.
Li Yan,Zhang Ting,Tian Wenjia,Hu Hejia,Xin Zengfeng,Ma Xiaojing,Ye Chenyi,Hang Kai,Han Xiuguo,Zhao Jie,Li Weixu
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Low back pain (LBP) is one of the most common complains in orthopedic outpatient department and intervertebral disc degeneration (IDD) is one of the most important reasons of LBP. The mechanisms of IDD contain a complex biochemical cascade which includes inflammation, vascular ingrowth, and results in degradation of matrix. In our study, we used both in vitro and in vivo models to investigate the relation between tissue inhibitor of metalloproteinase-3 (TIMP3) expression and IDD. Loss of TIMP3 expression was found in degenerative intervertebral disc (IVD), this change of expression was closely related with the dephosphorylation of smad2/3. Overexpression of TIMP3 significantly inhibited the release of TNF-α and matrix degradation induced by Lipopolysaccharide. Vascular ingrowth was also suppressed by TIMP3 in the in vitro and in vivo models. Further, animal experiments confirmed that the degeneration of IVD was reduced after overexpression of TIMP3 in nucleus pulposus. Taken together, our results indicated TIMP-3 might play an important role in the pathogenesis of IDD and therefore be a potential therapeutic target in the future.
10.1096/fj.201902364RR
Effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc.
Handa T,Ishihara H,Ohshima H,Osada R,Tsuji H,Obata K
Spine
STUDY DESIGN:This study is a unique in vitro study on the effects of hydrostatic pressure on human intervertebral disc metabolism. OBJECTIVE:To investigate the effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc. SUMMARY OF BACKGROUND DATA:Mechanical stress and hydrostatic pressures influence proteoglycan and protein synthesis rates in bovine articular cartilage and coccygeal discs. However, the mechanism of matrix synthesis regulation of the intervertebral disc under mechanical stress has not been elucidated. METHODS:Twenty-eight human lumbar intervertebral discs obtained from surgery and from cadavers at autopsy were used. Each tissue fraction was charged with medium in a plastic syringe and placed in a water-filled hydrostatic pressure-control vessel. The hydrostatic pressures applied were 1 (control), 3, and 30 atm (atm = atmospheres) for 2 hours. The proteoglycan and protein synthesis rates were determined by radioisotope incorporation. The production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 were measured by a one-step enzyme immunoassay method using monoclonal antibodies. RESULTS:Three atm pressure stimulated proteoglycan synthesis rates in the nucleus pulposus and inner anulus (n = 14 in each tissue). Compared with the control group, 30 atm pressure significantly inhibited proteoglycan synthesis in the inner anulus (P = 0.011). In the nucleus pulposus, matrix metalloproteinase-3 production was stimulated at a pressure of 30 atm relative to 3 atm (P = 0.014, n = 16 in each tissue). The highest tissue inhibitor of metalloproteinases-1 production showed highest values at 3 atm pressure in the inner anulus (n = 16 in each tissue). CONCLUSION:The results suggest that hydrostatic pressure influences intervertebral disc cell metabolism. A physiologic level of hydrostatic pressure (3 atm) may act as an anabolic factor for stimulation of proteoglycan synthesis and tissue inhibitor of metalloproteinases-1 production. This may be essential for maintaining the matrix of the disc. If the pressure was 30 atm or more or 1 atm or less, a catabolic effect will be predominant, with reduction of proteoglycan synthesis rate and increase of matrix metalloproteinase-3 production. Abnormal hydrostatic pressure, therefore, may accelerate disc degeneration.
IL-6/YAP1/β-catenin signaling is involved in intervertebral disc degeneration.
Chen Jian,Mei Zhengfeng,Huang Bao,Zhang Xuyang,Liu Junhui,Shan Zhi,Wang Jiasheng,Wang Xianjun,Zhao Fengdong
Journal of cellular physiology
Yes-associated protein 1 (YAP1) is a transcriptional coactivator and negative regulator of the Hippo pathway. It regulates diverse cellular processes, such as cell proliferation, contact inhibition, and tissue size. However, the role of YAP1 in intervertebral disc degeneration (IDD) remains elusive. Here, we demonstrated that YAP1 was activated by Interleukin 6 (IL-6) through tyrosine phosphorylation in nucleus pulposus cells (NP cells). Overexpression of YAP1 decreased Sox-9, Col-II, aggrecan expression, whereas increased matrix metalloproteinases 13 level. In contrast, knockdown of YAP1 by small interfering RNA (siRNA) showed opposite effects and rescued IL-6 induced NP cells degeneration. In addition, western blot showed that IL-6 treatment increased YAP1 and β-catenin protein level; co-immunoprecipitation (Co-IP) and immunofluorescence analysis showed that IL-6 enhanced YAP1 and β-catenin interaction and nuclear accumulation. Knockdown of β-catenin by siRNA blocked IL-6 treatment or YAP1 overexpression induced degeneration. Moreover, we found that verteporfin, a specific inhibitor of YAP1, effectively alleviated IDD development in rat disks. Taken together, our findings indicated that YAP1 plays an important role in IDD, and β-catenin is essential for IL-6/YAP1 signaling.
10.1002/jcp.27065
Matrix metalloproteinase 28, a novel matrix metalloproteinase, is constitutively expressed in human intervertebral disc tissue and is present in matrix of more degenerated discs.
Gruber Helen E,Ingram Jane A,Hoelscher Gretchen L,Zinchenko Natalia,Norton H James,Hanley Edward N
Arthritis research & therapy
INTRODUCTION:The regulation and elevation in expression of the catabolic matrix metalloproteinases (MMPs) is of high importance in the human intervertebral disc since upregulation of these matrix-degrading enzymes results in matrix destruction associated with disc degeneration. MMP28 (epilysin) is a newly discovered MMP believed to play a role in matrix composition and turnover in skin. It is present in basal keratinocytes where its expression is upregulated with wound repair, and in cartilage and synovium where it is upregulated in osteoarthritis. Recent work has shown that mechanical compression can act to modulate expression of MMP28. The expression of MMP28 is unexplored in the intervertebral disc. METHODS:Following approval by our human subjects institutional review board, we employed microarray analyses to evaluate in vivo expression of MMP28 and the MMP28 precursor in human disc tissue, and utilized immunohistochemistry to determine cellular and extracellular matrix localization of MMP28 in 35 human disc tissue specimens. The percentage of cells positive for MMP28 immunocytochemical localization was also determined. RESULTS:The present work documents the expression and presence of MMP28 in cells and extracellular matrix (ECM) of the human intervertebral disc. Gene expression levels in human disc tissue were detectable for both MMP28 and the MMP28 precursor. MMP28 cytoplasmic localization was present in cells of the outer annulus; it was also present in some, but not all, cells of the inner annulus and nucleus. MMP28 was not found in the ECM of healthier Grade I to II discs, but was identified in the ECM of 61% of the more degenerated Grade III to V discs (P = 0.0018). There was a significant difference in cellular MMP28 distribution in the disc (P = 0.008): the outer annulus showed the largest percentage of cells positive for MMP28 immunolocalization, followed by the inner annulus and then the nucleus. Herniated discs showed a significantly greater proportion of MMP28-positive cells compared with nonherniated discs (P = 0.034). CONCLUSIONS:Findings presented here show the first documentation of intervertebral disc expression and production of MMP28. MMP28 was found in both disc cell cytoplasm and in the ECM of more degenerated specimens, with greater cellular localization in the outer annulus and in herniated disc specimens. These findings are important because of the key role of MMPs in disc turnover and homeostasis, and previous indications of a role for this MMP in matrix repair and matrix turnover in other tissues. Our data, which show the presence of MMP28 in human disc tissue, suggest that MMP28 may have a potentially important role in ECM modulation in the healthy and degenerating disc.
10.1186/ar2876
TAK-715 alleviated IL-1β-induced apoptosis and ECM degradation in nucleus pulposus cells and attenuated intervertebral disc degeneration ex vivo and in vivo.
Arthritis research & therapy
BACKGROUND:Intervertebral disc degeneration (IDD) is one of the most common disorders related to the spine. Inflammation, apoptosis and extracellular matrix (ECM) degradation contribute to disc degeneration in nucleus pulposus cells (NPCs). This study focused on the role and mechanism of the p38 inhibitor TAK-715 in intervertebral disc degeneration. METHODS:NPCs were treated with IL-1β to mimic apoptosis, followed by the addition of TAK-715. It was determined that apoptosis, inflammatory mediators (COX-2), inflammatory cytokines (HMGB1), and ECM components (collagen II, MMP9, ADAMTS5, and MMP3) existed in NPCs. In addition, the p38MAPK signaling pathways were examined. The role of TAK-715 in vivo was determined by acupuncture-induced intervertebral disc degeneration. Following an intradiscal injection of TAK-715, MRI and a histopathological analysis were conducted to assess the degree of degeneration. RESULTS:IL-1β-induced apoptosis was alleviated by TAK-715 in vitro, and antiapoptotic proteins were upregulated. Furthermore, TAK-715 blocked IL-1β-induced inflammatory mediator production (COX-2) and inflammatory cytokine production (HMGB1) and degraded the ECM (collagen II, MMP9, ADAMTS5, and MMP3). By inhibiting the phosphorylation of p38, TAK-715 exerted its effects. In a rat tail model, TAK-715 ameliorates puncture-induced disc degeneration based on MRI and histopathology evaluations. CONCLUSION:TAK-715 attenuated intervertebral disc degeneration in vitro and in vivo, suggesting that it might be an effective treatment for IDD.
10.1186/s13075-023-03028-4
Extracellular vesicles derived from mesenchymal stem cells confer protection against intervertebral disc degeneration through a microRNA-217-dependent mechanism.
Osteoarthritis and cartilage
OBJECTIVE:Extracellular vesicles released by mesenchymal stem cells (MSC-EVs) can be applied to alleviate intervertebral disc degeneration (IVDD) by curbing apoptosis of nucleus pulposus cells (NPCs). The current study aims to evaluate the effect of MSC-EVs on NPC apoptosis and IVDD and the related regulatory mechanisms involving microRNA (miR)-217. METHOD:Expression of miR-217 was examined in tumor necrosis factor-α (TNF-α)-induced NPCs and MSC-EVs, followed by identification in the relationship between miR-217, enhancer of zeste homolog 2 (EZH2) and forkhead box O-3 (FOXO3). After isolation of EVs from MSCs and subsequent co-culture with NPCs, we assessed effects of miR-217 on NPC viability, autophagy, senescence and apoptosis along with extracellular matrix (ECM) degradation. Further in vivo experiments were conducted in rat models of IVDD to substantiate the effect of miR-217 on IVDD. RESULTS:Poor miR-217 expression was found in TNF-α-induced NPCs, while high miR-217 expression was identified in MSC-EVs (P < 0.05). MSC-EVs transferred miR-217 to NPCs and increased its expression, thus attenuating NPC apoptosis and ECM degradation (elevated collagen II and aggrecan but reduced MMP13 and ADAMTS5) (P < 0.05). miR-217 targeted EZH2, and EZH2 bound to the FOXO3 promoter and consequently downregulated its expression. FOXO3 restrained NPC apoptosis and ECM degradation by stimulating cell autophagy (P < 0.05). Furthermore, in vivo experimental results confirmed the suppressive role of miR-217 shuttled by MSC-EVs in IVDD. CONCLUSION:Overall, the delivery of miR-217 may be a novel mechanism underlying the effect of MSC-EVs on NPC apoptosis and ECM degradation following IVDD.
10.1016/j.joca.2022.08.009
Wogonin mitigates intervertebral disc degeneration through the Nrf2/ARE and MAPK signaling pathways.
Fang Weijing,Zhou Xiaopeng,Wang Jingkai,Xu Langhai,Zhou Lijuan,Yu Wei,Tao Yiqing,Zhu Jian,Hu Bin,Liang Chengzhen,Li Fangcai,Hua Jianming,Chen Qixin
International immunopharmacology
Intervertebral disc degeneration (IVDD) is a prevalent disease characterized by the progressive loss of the extracellular matrix in the local nucleus pulposus region. Metalloproteinases and pro-inflammatory cytokines play an important role in this process. Thus, anti-inflammatory strategies are an important component of IVDD treatment. Wogonin, a naturally existing monoflavonoid, has been reported to have potential anti-inflammatory effects in some inflammatory diseases. Hence, in our present study we investigated the protective effects and potential mechanisms of wogonin in rat nucleus pulposus cells that had been treated with interleukin-1beta (IL-1β) to induce severe IVDD. An in vivo rat caudal vertebrae needle-stab model was also designed and its validity was evaluated as an IVDD model. The results demonstrated that wogonin suppressed IL-1β-induced inflammatory mediators (iNOS, IL-6 and COX2) and matrix-degrading proteinases (MMP1, MMP3, MMP13 and ADAMTS4). Wogonin also upregulated some of the key components of the extracellular matrix, such as collagen II. Furthermore, we discovered that wogonin exerted anti-inflammatory effects by activating the Nrf2/HO-1-SOD2-NQO1-GCLC signaling axis. Moreover, the IL-1β-induced stimulation of the MAPK signaling pathway was reversed by wogonin treatment. The in vivo MRI and histological results also revealed that wogonin protected the nucleus pulposus from the progression of IVDD. Therefore, wogonin may be a potential agent for the treatment of IVDD.
10.1016/j.intimp.2018.10.024
The potential role and trend of HIF‑1α in intervertebral disc degeneration: Friend or foe? (Review).
Li Yongjin,Liu Shen,Pan Dayu,Xu Baoshan,Xing Xuewu,Zhou Hengxing,Zhang Bin,Zhou Suzhe,Ning Guangzhi,Feng Shiqing
Molecular medicine reports
Lower back pain (LBP) is one of the most common reasons for seeking medical advice in orthopedic clinics. Increasingly, research has shown that symptomatic intervertebral disc degeneration (IDD) is mostly related to LBP. This review first outlines the research and findings of studies into IDD, from the physiological structure of the intervertebral disc (IVD) to various pathological cascades. The vicious cycles of IDD are re‑described in relation to the analysis of the relationship among the pathological mechanisms involved in IDD. Interestingly, a 'chief molecule' was found, hypoxia‑inducible factor‑1α (HIF‑1α), that may regulate all other mechanisms involved in IDD. When the vicious cycle is established, the low oxygen tension activates the expression of HIF‑1α, which subsequently enters into the hypoxia‑induced HIF pathways. The HIF pathways are dichotomized as friend and foe pathways according to the oxygen tension of the IVD microenvironment. Combined with clinical outcomes and previous research, the trend of IDD development has been predicted in this paper. Lastly, an early precautionary diagnosis and treatment method is proposed whereby nucleus pulposus tissue for biopsy can be obtained through IVD puncture guided by B‑ultrasound when the patient is showing symptoms but MRI imaging shows negative results. The assessment criteria for biopsy and the feasibility, superiority and challenges of this approach have been discussed. Overall, it is clear that HIF‑1α is an indispensable reference indicator for the accurate diagnosis and treatment of IDD.
10.3892/mmr.2021.11878
Efficiency of dual siRNA-mediated gene therapy for intervertebral disc degeneration (IVDD).
Banala Rajkiran Reddy,Vemuri Satish Kumar,Dar Ghulam Hassan,Palanisamy Vijayanand,Penkulinti Murahari,Surekha M V,Gurava Reddy A V,Nalam Madhusudhana Rao,Subbaiah Gpv
The spine journal : official journal of the North American Spine Society
BACKGROUND CONTEXT:One of the common causes of low back pain is intervertebral disc degeneration. The pathophysiology of disc degeneration involves apoptosis of nucleus pulposes cells and degradation of extra cellular matrix (ECM). Caspase 3 plays a central role in apoptosis and the ADAMTS5 (A Disintegrin and Metalloproteinase with Thrombospondin motifs 5) gene plays a critical role in ECM degradation. Hence, we hypothesized that if one can silence these two genes, both apoptosis and ECM degradation can be prevented, thereby preventing the progression and even reverse disc degeneration. PURPOSE:The purpose of this study is to demonstrate the regenerative potential of small interfering RNA (siRNA) designed against Caspase 3 and ADAMTS5 genes in an in vitro and animal model of disc degeneration. STUDY DESIGN:In vitro study followed by in vivo study in a rabbit model. METHODS:In vitro studies were done using the human hepatocellular carcinoma (Hep G2) cell line for validating the efficacy of liposomal siRNA in controlling the expression of genes (Caspase 3 and ADAMTS5). Later, siRNA's validation was done in a rabbit annular punctured model by administering siRNA's individually (Caspase 3 and ADAMTS5) and in combination Caspase3-ADAMTS5) for assessing their synergistic effect in down regulating the gene expression in the degenerative discs. Annular punctured intervertebral discs of the rabbit were injected with siRNA formulations (single and dual) and phosphate buffer saline, one week after initial puncture. Magnetic resonance imaging (MRI) scans were done before and after siRNA treatment (1, 4 and 8 weeks) for assessing the progression of disc degeneration. The histopathology and real time polymerase chain reaction (RT-PCR) studies were done for evaluating their efficacy. We did not receive any funding for conducting the study, and we do not have a conflict of interest with any researchers or scientific groups. RESULTS:The observations made from both in vitro and in vivo studies indicate the beneficial effects of siRNA formulation in down regulating the expression of Caspase 3 and ADAMTS5 genes. The MRI and histopathological evaluation showed that the disc degeneration was progressive in phosphate buffer saline and AT5-siRNA injected discs but the discs that received Caspase 3-siRNA and dual siRNA (Cas3-AT5-siRNA) formulation showed signs of recovery and regeneration 4 and 8 weeks after injection. The efficacy of siRNA designed against Cas3 and AT5 was also assessed in both in vitro and in vivo experiments by using RT-PCR analysis and the results showed downregulation of Caspase 3 gene in Caspase 3-siRNA group, but there was no significant downregulation of ADAMTS5 gene in ADAMTS5-siRNA group (ie, indicated by fold change). Synergistic effect was observed in the group that received dual siRNA (Cas3-AT5 siRNA) formulation. CONCLUSIONS:This experiment suggests that intervention by siRNA treatment significantly reduced the extent of apoptosis in the discs. CLINICAL SIGNIFICANCE:Delivery of siRNA directly into spinal discs has a potential in treating disc degeneration nonsurgically.
10.1016/j.spinee.2018.10.016
Kinetics of tissue and serum matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 in intervertebral disc degeneration and disc herniation.
Nishida T
The Kurume medical journal
We have evaluated the correlation between disc herniation or degeneration and the levels of matrix metalloproteinase-3 (MMP-3) or tissue inhibitor of metalloproteinases-1 (TIMP-1). The lumbar discs obtained from 22 autopsied cadavers and 21 patients with lumbar disc herniation were examined. Immunohistochemical study: The number of MMP-3-producing and TIMP-1-producing cells increased as disc herniation or degeneration progressed. Zymography: Activated type MMP-3 was observed in the normal and herniated intervertebral disc excluding the intervertebral disc with protrusion-type herniation. RT-PCR: The MMP-3 gene was expressed in all groups. However, the expression of the TIMP-1 gene was weak in the normal group as well as in the protrusion type disc herniation. Determination of tissue and serum levels of MMP-3 and TIMP-1 was increased as disc degeneration or herniation progressed. There were positive correlations between the number of MMP-3-producing or TIMP-1-producing cells in the intervertebral disc and the tissue or serum levels of MMP-3 and TIMP-1. These results suggested that MMP-3 and TIMP-1 were closely related to the progression of disc degeneration or herniation. The results also suggested that MMP-3 and TIMP-1 exhibited similar kinetics. Moreover, the serum levels of MMP-3 and TIMP-1 reflected the progression of disc degeneration and herniation.
Accumulated Spinal Axial Biomechanical Loading Induces Degeneration in Intervertebral Disc of Mice Lumbar Spine.
Lao Yang-Jun,Xu Tao-Tao,Jin Hong-Ting,Ruan Hong-Feng,Wang Ji-Tao,Zhou Li,Wang Ping-Er,Wang Jian,Ying Jun,Zhang Yuan-Bin,Luo Cheng,Fu Fang-da,Tong Pei-Jian,Xiao Lu-Wei,Wu Cheng-Liang
Orthopaedic surgery
OBJECTIVE:To investigate the effect of accumulated spinal axial biomechanical loading on mice lumbar disc and the feasibility of applying this method to establish a mice intervertebral disc degeneration model using a custom-made hot plate cage. In previous studies, we observed that the motion pattern of mice was greatly similar to that of humans when they were standing and jumping on their lower limbs. There is little data to demonstrate whether or not accumulated spinal axial biomechanical loading could induce intervertebral disc degeneration in vivo. METHODS:Twenty-four 0-week-old mice were randomly divided into model 1-month and 3-month groups, and control 1-month and 3-month groups (n = 6 per group). The model groups was transferred into the custom-made hot plate cage three times per day for modeling. The control group was kept in a regular cage. The intervertebral disc samples of the L -L were harvested for histologic, molecular, and immunohistochemical studies after modeling for 1 and 3 months. RESULTS:Accumulated spinal axial biomechanical loading affects the histologic, molecular, and immunohistochemical changes of mice L L intervertebral discs. Decreased height of disc and endplate, fissures of annulus fibrosus, and ossification of cartilage endplate were found in morphological studies. Immunohistochemical studies of the protein level showed a similar expression of type II collagen at 1 month, but a slightly decreased expression at 3 months, and an increased expression level of type X collagen and matrix metalloproteinase 13 (MMP13). Molecular studies showed that ColIIa1 and aggrecan mRNA expression levels were slightly increased at 1 month (P > 0.05), but then decreased slightly (P > 0.05). ColXa1, ADAMTS-5, and MMP-13 expression levels werer increased both at 1 and 3 months (P < 0.05). In addition, increased expression of Runx2 was observed. CONCLUSION:Accumulated spinal axial loading provided by a custom-made hot plate accelerated mice lumbar disc and especially endplate degeneration. However, this method requires further development to establish a lumbar disc degeneration model.
10.1111/os.12365
Systemic clearance of p16 -positive senescent cells mitigates age-associated intervertebral disc degeneration.
Aging cell
RATIONALE:Age-related changes in the intervertebral discs are the predominant contributors to back pain, a common physical and functional impairment experienced by older persons. Cellular senescence, a process wherein cells undergo growth arrest and chronically secrete numerous inflammatory molecules and proteases, has been reported to cause decline in the health and function of multiple tissues with age. Although senescent cells have been reported to increase in intervertebral degeneration (IDD), it is not known whether they are causative in age-related IDD. OBJECTIVE:The study aimed to elucidate whether a causal relationship exists between cellular senescence and age-related IDD. METHODS AND RESULTS:To examine the impact of senescent cells on age-associated IDD, we used p16-3MR transgenic mice, which enables the selective removal of p16 -positive senescent cells by the drug ganciclovir. Disc cellularity, aggrecan content and fragmentation alongside expression of inflammatory cytokine (IL-6) and matrix proteases (ADAMTS4 and MMP13) in discs of p16-3MR mice treated with GCV and untreated controls were assessed. In aged mice, reducing the per cent of senescent cells decreased disc aggrecan proteolytic degradation and increased overall proteoglycan matrix content along with improved histological disc features. Additionally, reduction of senescent cells lowered the levels of MMP13, which is purported to promote disc degenerative changes during aging. CONCLUSIONS:The findings of this study suggest that systemic reduction in the number of senescent cells ameliorates multiple age-associated changes within the disc tissue. Cellular senescence could therefore serve as a therapeutic target to restore the health of disc tissue that deteriorates with age.
10.1111/acel.12927
The imbalance between TIMP3 and matrix-degrading enzymes plays an important role in intervertebral disc degeneration.
Li Yan,Li Kang,Han Xiuguo,Mao Chuanyuan,Zhang Kai,Zhao Tengfei,Zhao Jie
Biochemical and biophysical research communications
It is well-known that one of the most important features of intervertebral disc degeneration (IDD) is the extracellular matrix (ECM) degradation. Collagen and aggrecan are major components of ECM; the degradation of ECM in intervertebral discs (IVDs) is closely related to the activities of collagenase and aggrecanase. TIMP-3 is the most efficient inhibitor of aggrecanase in IVD. However, only few studies focus on the potential relationship between TIMP-3 and IDD. In our study, we found TIMP-3 gene expression was decreased after stimulating with LPS in rat nucleus pulposus (NP) cells. Then we used a lentivirus vector to reconstruct rat NP cells which high expressed TIMP-3 gene (LV-TIMP3). The upregulation of MMPs and ADAMTSs induced by LPS was significantly inhibited in LV-TIMP3 cells. After overexpression of TIMP-3, the aggrecan breakdown caused by LPS was also reduced in both monolayer culture and three-dimension culture model. To further study the relation between TIMP-3 and IDD, we collected human NP tissue samples of different degenerative degrees. Real-time PCR and immunohistochemical staining showed that the expression of TIMP-3 was negatively correlated with the degree of intervertebral disc degeneration, while MMP-1 and ADAMTS-4 were markedly increased in degenerative IVD. Taken together, our results suggest that the imbalance between aggrecanase and TIMP-3 may play an important role in the pathogenesis of IDD and therefore be a potential therapeutic target for treating IDD.
10.1016/j.bbrc.2015.12.020
Analysis of genetic polymorphisms associated with intervertebral disc degeneration.
Kitis Serkan,Coskun Zeynep Mine,Tasdemir Pelin,Tuncez Ebru,Zamani Ayse Gul,Acar Aynur
Cellular and molecular biology (Noisy-le-Grand, France)
Intervertebral disc degeneration (IVDD) is a common degenerative spinal condition. Recent studies have shown that the incidence of disc herniation and disc degeneration may be explained by genetic factors. In this study, we investigated the link between various polymorphic variants of the vitamin D receptor (VDR), matrix metalloproteinase 2 (MMP2), and insulin like growth factor 1 receptor (IGF1R) genes and IVDD in patients with IVDD, in Turkey. We examined and genotyped 199 patients with IVDD and 197 healthy individuals. Genomic DNA was isolated from the peripheral blood leukocytes of all participants, and analyzed using real-time PCR. Via melting curve analysis, VDR, MMP2, and IGF1R polymorphism variant distributions were determined. The patients with IVDD showed higher frequencies of the VDR ApaI A allele genotype as compared to the control group; however, there were no significant differences in the frequencies or allelic distributions of the IGF1R and MMP2 genotypes between the IVDD patients and the control group. The incidence of IVDD in these Turkish patients is correlated with the VDR ApaI gene polymorphism, but not with the IGF1R and MMP2 polymorphisms.
Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration.
Vo Nam V,Hartman Robert A,Yurube Takashi,Jacobs Lloydine J,Sowa Gwendolyn A,Kang James D
The spine journal : official journal of the North American Spine Society
BACKGROUND CONTEXT:Destruction of extracellular matrix (ECM) leads to intervertebral disc degeneration (IDD), which underlies many spine-related disorders. Matrix metalloproteinases (MMPs), and disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) are believed to be the major proteolytic enzymes responsible for ECM degradation in the intervertebral disc (IVD). PURPOSE:To summarize the current literature on gene expression and regulation of MMPs, ADAMTSs, and tissue inhibitors of metalloproteinases (TIMPs) in IVD aging and IDD. METHODS:A comprehensive literature review of gene expression of MMP, ADAMTS, and TIMP in human IDD and reported studies on regulatory factors controlling their expressions and activities in both human and animal model systems. RESULTS:Upregulation of specific MMPs (MMP-1, -2, -3, -7, -8, -10, and -13) and ADAMTS (ADAMTS-1, -4, and -15) were reported in human degenerated IVDs. However, it is still unclear from conflicting published studies whether the expression of ADAMTS-5, the predominant aggrecanase, is increased with IDD. Tissue inhibitors of metalloproteinase-3 is downregulated, whereas TIMP-1 is upregulated in human degenerated IVDs relative to nondegenerated IVDs. Numerous studies indicate that the expression levels of MMP and ADAMTS are modulated by a combination of many factors, including mechanical, inflammatory, and oxidative stress, some of which are mediated in part through the p38 mitogen-activated protein kinase pathway. Genetic predisposition also plays an important role in determining gene expression of MMP-1, -2, -3, and -9. CONCLUSIONS:Upregulation of MMP and ADAMTS expression and enzymatic activity is implicated in disc ECM destruction, leading to the development of IDD. Future IDD therapeutics depends on identifying specific MMPs and ADAMTSs whose dysregulation result in pathological proteolysis of disc ECM.
10.1016/j.spinee.2012.02.027
Biochemical composition and turnover of the extracellular matrix of the normal and degenerate intervertebral disc.
Sivan Sarit Sara,Hayes Anthony J,Wachtel Ellen,Caterson Bruce,Merkher Yulia,Maroudas Alice,Brown Sharon,Roberts Sally
European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society
BACKGROUND:The intervertebral disc (IVD) is a complex cartilaginous structure which functions to resist biomechanical loads during spinal movement. It consists of the highly viscous cartilaginous nucleus pulposus, which is surrounded laterally by a thick outer ring of fibrous cartilage-the annulus fibrosus-and sandwiched inferiorly and superiorly by the cartilage end-plates. The main extracellular matrix molecules of the disc are collagens, proteoglycans, glycoproteins and elastin. The disc also contains appreciable amounts of water, matrix-degrading protease enzymes and their inhibitors, soluble signalling molecules and various metabolic breakdown products. METHODS:This review provides a comprehensive description of the biochemical composition of the extracellular matrix of the IVD and, specifically, the proteases involved in its molecular turnover. Quantitation of the turnover rates using racemization of aspartic acid as a molecular clock is also discussed. CONCLUSIONS:Molecular turnover rates of the major constituent matrix macromolecules of the IVD are found to be particularly slow, especially in the case of collagen. Over a normal human life span, this slow turnover may compromise the structural integrity of the IVD extracellular matrix essential for normal physiological functioning.
10.1007/s00586-013-2767-8
Elevated expression of hypoxia-inducible factor-2alpha regulated catabolic factors during intervertebral disc degeneration.
Life sciences
HEADINGS AIMS:The present study determined whether nucleus pulposus (NP) cells express hypoxia-inducible factor-2alpha (HIF-2α) and assessed its role in regulating the expression of catabolic factors during intervertebral disc degeneration. MATERIALS AND METHODS:Human degenerated NP tissues were acquired to examine the HIF-2α expression levels using immunohistochemistry, western blotting, and Real-time PCR. Human NP cells were cultivated under normoxic or hypoxic conditions, and the HIF-2α expression was determined. Then, human NP cells were treated with HIF-2α plasmids, HIF-2α siRNA, and tumor necrosis factor-alpha (TNF-α) to evaluate the role of HIF-2α in regulating matrix metalloproteinase (MMP) and aggrecanase expression. An in vivo rabbit disc degeneration model was established to demonstrate that HIF-2α plays a critical role in disc degeneration. KEY FINDINGS:We found that HIF-2α had a markedly elevated expression in human degenerated discs in the Grade III stage. HIF-2α protein and gene transcript levels in vitro were relatively higher under hypoxic conditions. The expression of MMP-13, ADAMTS-4 was decreased significantly in HIF-2α silencing condition, while the over-expression resulted in significantly increased levels of MMP-13 and ADAMTS-4. When cytokine TNF-α was added, HIF-2α was induced by nuclear factor-κB (NF-κB). The in vivo experiments showed that the HIF-2α controlled the catabolic factors MMP-13 and ADAMTS-4 that regulated the collagen II and aggrecan metabolism in disc degeneration. SIGNIFICANCE:HIF-2α is a catabolic regulator in disc degeneration and directly controls the catabolic genes. The suppression of HIF-2α expression leads to decelerates extracellular matrix degradation that might represent a therapeutic target for the degenerative disc.
10.1016/j.lfs.2019.116565
Effects of suppressing bioavailability of insulin-like growth factor on age-associated intervertebral disc degeneration.
JOR spine
Suppression of the insulin-like growth factor-1 (IGF-1) signaling pathway reduces age-related disorders and increases lifespan across species, making the IGF-1 pathway a key regulator of aging. Previous in vitro intervertebral disc cell studies have reported the pro-anabolic effect of exogenously adding IGF-1 on matrix production. However, the overall effects of suppressing IGF-1 signaling on age-related intervertebral disc degeneration (IDD) is not known. Here, the effects of suppressing IGF-1 signaling on age-related IDD in vivo were examined using mice. These are animals with targeted deletion of pregnancy-associated plasma protein A (PAPPA), the major protease that cleaves inhibitory IGF binding proteins that control bioavailability of IGF-1 for cell signaling. Compared to age-matched wild-type (Wt) littermates, reduced levels of matrix proteoglycan (PG) and aggrecan were seen in discs of 23-month old mice. Decreased aggrecanolysis and expression of two key catabolic markers, matrix metalloproteinase-3 and a disintegrin and metalloproteinase with thrombospondin motifs-4, were also observed in discs of old mice compared to Wt littermates. Suppressing IGF-1 signaling has been implicated to shift cellular metabolism toward maintenance rather than growth and decreasing cellular senescence. Along this line, discs of old mice also exhibited lower cellular senescence, assessed by p53 and lamin B1 markers. Collectively, the data reveal complex regulation of disc matrix homeostasis by PAPPA/IGF-1 signaling during chronologic aging, that is, reduced IGF-1 bioavailability confers the benefit of decreasing disc cellular senescence and matrix catabolism but also the disadvantage of decreasing disc PG matrix anabolism. This pathway requires further mechanistic elucidation before IGF-1 could be considered as a therapeutic growth factor for treating IDD.
10.1002/jsp2.1112
Intervertebral disc degeneration and ectopic bone formation in apolipoprotein E knockout mice.
Zhang Dawei,Jin Li,Reames Davis L,Shen Francis H,Shimer Adam L,Li Xudong
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Cardiovascular risk factors are known to be associated with intervertebral disc degeneration, but the underlying mechanism is still unclear. The ApoE knockout (KO) mouse is a well-established model for atheroscelorosis. We hypothesized that ApoE is involved in maintaining disc health and that ApoE KO mice will develop early disc degeneration. Discs of ApoE KO and wild-type (WT) mice were characterized with histological/immunological, biochemical, and real-time RT-PCR assays. A comparison of the extracellular matrix production was also performed in disc cells. We demonstrated that ApoE was highly expressed in the endplates of WT discs, and ectopic bone formed in the endplates of ApoE KO discs. Glycosaminoglycan content was decreased in both ApoE KO annulus fibrosus (AF) and nucleus pulposus (NP) cells. Collagen levels were increased in AF and decreased in NP cells. Matrix metalloproteinase-3, -9, and -13 expressions were increased, which may partially explain the impaired matrix production. We also found collagen I, II, aggrecan, and biglycan mRNA expressions were increased in AF cells but decreased in NP cells. Apoptosis was increased in the ApoE KO NP tissue. These results suggest early disc degeneration changes in the ApoE KO mice. ApoE may play a critical role in disc integrity and function.
10.1002/jor.22216
Syndecan-4 in intervertebral disc and cartilage: Saint or synner?
Binch Abbie L A,Shapiro Irving M,Risbud Makarand V
Matrix biology : journal of the International Society for Matrix Biology
The ECM of the intervertebral disc and articular cartilage contains a highly organised network of collagens and proteoglycans which resist compressive forces applied to these tissues. A pathological hallmark of the intervertebral disc is the imbalance between production of anabolic and catabolic factors by the resident cells. This process is thought to be mediated by pro-inflammatory cytokines, predominantly TNF-α and IL-1β, which upregulate expression of matrix degrading enzymes such as MMPs and ADAMTSs. This imbalance ultimately results in tissue degeneration causing failure of the biomechanical function of the tissues. A similar cascade of events is thought to occur in articular cartilage during development of osteoarthritis. Within these skeletal tissues a small, cell surface heparan sulphate proteoglycan; syndecan-4 (SDC4) has been implicated in maintaining physiological functions. However in the degenerating niche of the intervertebral disc and cartilage, dysregulated activities of this molecule may exacerbate pathological changes. Studies in recent years have elucidated a role for SDC4 in mediating matrix degradation in both intervertebral discs and cartilage by controlling ADAMTS-5 function and MMP3 expression. Discourse presented in this review highlights the potential of SDC4 as a possible therapeutic target in slowing the progression of ECM degradation in both degenerative disc disease and osteoarthritis.
10.1016/j.matbio.2016.01.005
Wnt5a suppresses inflammation-driven intervertebral disc degeneration via a TNF-α/NF-κB-Wnt5a negative-feedback loop.
Li Z,Zhang K,Li X,Pan H,Li S,Chen F,Zhang J,Zheng Z,Wang J,Liu H
Osteoarthritis and cartilage
OBJECTIVE:This study was to investigate the molecular role of Wnt5a on inflammation-driven intervertebral disc degeneration (IVDD). METHODS:The expression of Wnt5a was analyzed in human nucleus pulposus (NP) tissues with immunohistochemical staining. The effects of Wnt5a on matrix production were assessed by RT-qPCR and western blotting. Small interfering RNAs (siRNAs), promoter deletion assay, and promoter binding site mutant were used to reveal the molecular role of Wnt5a in TNF-α-induced matrix metalloproteinase (MMP) expression. The regulatory effects of TNF-α on Wnt5a were investigated with pharmachemical inhibitors and siRNA experiment. RESULTS:The expression of Wnt5a was elevated in moderately degenerated human NP tissue with similar expression pattern of TNF-α. In NP cells, Wnt5a significantly increased aggrecan and collagen II expression. Inhibition of JNK or interfering Sox9 gene expression significantly suppressed Wnt5a-induced matrix production. AP-1(JunB) binding sites were located in Sox9 promoter and mutation of these sites sabotaged Wnt5a-induced Sox9 up-regulation and subsequent matrix genes expression. Notably, Wnt5a, which was induced by TNF-α, on the other way round suppressed TNF-α-NF-κB (p65) signaling and subsequent MMPs expression. In vivo studies with MR imaging confirmed the protective role of Wnt5a in IVDD. CONCLUSIONS:Wnt5a, which can be induced by TNF-α, increased matrix production in a Sox9-dependent manner through the activation of JNK-AP1 (JunB) signaling, and antagonized TNF-α-induced up-regulation of MMPs through the inhibition of NF-κB signaling. It indicates that Wnt5a suppresses IVDD through a TNF-α/NF-κB-Wnt5a negative-feedback loop.
10.1016/j.joca.2018.04.002
Matrix metalloproteinase-26, a novel MMP, is constitutively expressed in the human intervertebral disc in vivo and in vitro.
Gruber Helen E,Hoelscher Gretchen L,Ingram Jane A,Hanley Edward N
Experimental and molecular pathology
Matrix metalloproteinase (MMP) regulation and expression is important in the aging/degenerating human intervertebral disc. MMP-26 (also known as matrilysin-2 or endometase) is a newly discovered MMP which degrades type IV collagen, fibronectin, fibrinogen, vitronectin, denatured collagen types I-IV, insulin-like growth factor binding protein 1, and activated pro-MMP-9. Our objective here was to determine if it is present in human disc tissue and cultured disc cells. Immunohistochemistry and microarray gene expression analyses were used to evaluate the presence of MMP-26 in human disc tissue from healthy and degenerated discs. Immunohistochemistry was also applied to human annulus cells cultured in a collagen sponge. Cellular and matrix localization of MMP-26 was identified in the outer and inner annulus and in the nucleus pulposus. Fewer cells showed localization in the inner vs. outer annulus, and localization was sparse in the nucleus. During in vitro culture of annulus cells, MMP-26 was also expressed. Molecular analyses showed significant downregulation of expression of MMP-26 (p=0.03), and significant 9.8-fold upregulation of TGF-beta (p=0.01) in more degenerated discs vs. healthier discs. Findings document the first identification of MMP-26 in the disc at the molecular and protein levels. Results point to the potentially important role of MMP-26 in matrix modulation during disc health and degeneration.
10.1016/j.yexmp.2011.09.008
Sirtuin 6 prevents matrix degradation through inhibition of the NF-κB pathway in intervertebral disc degeneration.
Kang Liang,Hu Jia,Weng Yuxiong,Jia Jie,Zhang Yukun
Experimental cell research
Intervertebral disc degeneration (IDD) is marked by imbalanced metabolism of the extracellular matrix (ECM) in the nucleus pulposus (NP) of intervertebral discs. This study aimed to determine whether sirtuin 6 (SIRT6), a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases, protects the NP from ECM degradation in IDD. Our study showed that expression of SIRT6 markedly decreased during IDD progression. Overexpression of wild-type SIRT6, but not a catalytically inactive mutant, prevented IL-1β-induced NP ECM degradation. SIRT6 depletion by RNA interference in NP cells caused ECM degradation. Moreover, SIRT6 physically interacted with nuclear factor-κB (NF-κB) catalytic subunit p65, transcriptional activity of which was significantly suppressed by SIRT6 overexpression. These results suggest that SIRT6 prevented NP ECM degradation in vitro via inhibiting NF-κB-dependent transcriptional activity and that this effect depended on its deacetylase activity.
10.1016/j.yexcr.2017.02.023
Intervertebral Disc Degeneration and Low Back Pain Depends on Duration and Magnitude of Axial Compression.
Oxidative medicine and cellular longevity
Purpose:The pathological role of axial stress in intervertebral disc degeneration (IDD) is controversial, and there was no quantified study until now. Here, we tried to clarify the correlation between IDD or low back pain (LBP) and axial stress at different duration and magnitude in vitro and in vivo. Method:In vitro, the gene expression of aggrecan, matrix metalloproteinase-3 (MMP3), calcitonin gene-related peptide (CGRP), and substance P (SP) was measured when nucleus pulposus cells (NPCs) were compressed under gradual severity. In vivo, a measurable Ilizarov-type compression apparatus was established for single coccygeal (Co) intervertebral disc (IVD) compression of Co7-8 in mouse. Gradient stress was placed at 0.4 Mpa (mild), 0.8 Mpa (moderate), and 1.2 Mpa (severe) for three days to investigate the effect of the magnitude of axial stress. Additionally, mild compression with 3, 7, and 14 days was used to determine the effect of the duration of axial stress. Subsequently, we evaluated the severity of IDD and LBP by radiological X-ray film; histological examination with H&E staining; immunohistochemical analysis with collagen II, aggrecan, and CGRP staining; and western blot analysis with collagen II, aggrecan, MMP-3, and interleukin-1 (IL-1). Results:When NPCs suffered gradual increased mechanical stress, the cells exhibited gradual downregulated expression of extracellular matrix (ECM)-related gene of aggrecan, upregulated expression of IDD-related gene of MMP3, and LBP-related gene of CGRP and SP. In the meantime, with different magnitudes of axial stress, the IVD showed progressively severe IDD and LBP, with gradual narrowing intervertebral height, destruction of IVD anatomy, decreased ECM, and increased catabolic factors and proalgesic peptides. Conclusion:Axial compression is one of the critical pathological factors to cause IDD and LBP, and there was a strong positive correlation depended on the duration and magnitude of compression.
10.1155/2022/1045999
PTEN promotes intervertebral disc degeneration by regulating nucleus pulposus cell behaviors.
Xi Yongming,Ma Jinfeng,Chen Yan
Cell biology international
Intervertebral disc degeneration (IDD) is induced by multiple factors including increased apoptosis, decreased survival, and reduced extracellular matrix (ECM) synthesis in the nucleus pulposus (NP) cells. The tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is the only known lipid phosphatase counteracting the PI3K/AKT pathway. Loss of PTEN leads to activated PI3K/AKT signaling, which plays a key role in a variety of cancers. However, the role of PTEN/PI3K/AKT signaling nexus in IDD remains unknown. Here, we report that PTEN is overexpressed in degenerative NP, which correlates with inactivated AKT. Using the PTEN knockdown approach by lentivirus-mediated short interfering RNA gene transfer technique, we report that PTEN decreases survival but induces apoptosis and senescence of NP cells. PTEN also inhibits expression and production of ECM components including collagen II, aggrecan, and proteoglycan. Furthermore, PTEN modulates the expression of ECM regulatory molecules SOX-9 and matrix metalloproteinase-3 (MMP-3). Using small-molecule AKT inhibitor GDC-0068, we confirm that PTEN regulates NP cell behaviors through its direct targeting of PI3K/AKT. These findings demonstrate for the first time that PTEN/PI3K/AKT signaling axis plays an important role in the pathogenesis of IDD. Targeting PTEN using gene therapy may represent a promising therapeutic approach against disc degenerative diseases.
10.1002/cbin.11258
A Mouse Intervertebral Disc Degeneration Model by Surgically Induced Instability.
Oichi Takeshi,Taniguchi Yuki,Soma Kazuhito,Chang Song Ho,Yano Fumiko,Tanaka Sakae,Saito Taku
Spine
STUDY DESIGN:An experimental study to develop a mouse model of lumbar intervertebral disc degeneration (IDD). OBJECTIVE:The aim of this study was to develop a mouse lumbar IDD model using surgically induced instability and to compare the findings of this model to those in human IDD. SUMMARY OF BACKGROUND DATA:Previously, various kinds of inducers have been used to reproduce IDD in experimental animals; however, there is yet no standard mouse lumbar IDD model without direct injury to intervertebral disc. METHODS:A total number of 59 C57BL/6J male mice at 8 weeks old were used. Instability of lumbar spine was induced by surgical resection of posterior elements, including facet joints, supra- and interspinous ligaments. We then analyzed time course changes in radiographical (n = 17) and histological analyses (n = 42), and compared these findings with those in human IDD. RESULTS:Radiographical analyses showed that the disc height began to decrease in the first 2 weeks after the surgery, and the decrease continued throughout 12 weeks. Bone spurs at the vertebral rims were observed in the late stage of 8 and 12 weeks after the surgery. Histological analyses showed that the disorder of the anterior anulus fibrosus (AF) was initially obvious, followed by posterior shift and degeneration of the nucleus pulposus (NP). Proteoglycan detected in inner layer of AF and periphery of NP was decreased after 8 weeks. Immunohistochemistry displayed the increase of type I and X collagen, and matrix metalloproteinase 13 in the anterior AF. CONCLUSION:Surgical resection of posterior elements of mouse lumbar spine resulted in reproducible IDD. Because the present procedure does not employ direct injury to intervertebral disc and the radiological and histological findings are compatible with those in human IDD, it may contribute to further understanding of the native pathophysiology of IDD in future. LEVEL OF EVIDENCE:N/A.
10.1097/BRS.0000000000002427
Matrix metalloproteinases in the human intervertebral disc: role in disc degeneration and scoliosis.
Crean J K,Roberts S,Jaffray D C,Eisenstein S M,Duance V C
Spine
STUDY DESIGN:Biochemical study of human intervertebral discs collected at surgery from patients with low back pain associated with disc degeneration or scoliosis. Matrix metalloproteinases were studied by quantitative zymography. OBJECTIVE:To determine whether changes in the expression of matrix metalloproteinases will bring about tissue remodelling that contributes to the progressive nature and pathology of these diseases of the intervertebral disc. SUMMARY OF BACKGROUND DATA:The diseases of the intervertebral disc, degenerative disc disease and scoliosis, are both characterized by changes in the extracellular matrix components that will affect the mechanical function of the tissue. Matrix metalloproteinases are known to have the capability of degrading all the known extracellular matrix components of the disc. METHODS:Matrix metalloproteinases 2 and 9 were detected by gelatin-gel zymography and quantified by laser scanning densitometry. Both pro and active forms of the enzymes were measured. Thirty-four discs from patients with low back pain and 29 from patients with scoliosis were investigated. RESULTS:A correlation was found between the increasing levels of matrix metalloproteinases 2 and 9 and the grade of degenerative disc disease. In addition, the levels of these enzymes show a differential expression across the scoliotic disc with the highest levels in samples taken from the convexity of the curve. CONCLUSIONS:The difference between the concave and convex side of the scoliotic curve indicates that mechanical loads might influence the expression of these enzymes. The increased expression of these enzymes in both degenerative disc disease and scoliosis strongly suggests that they may affect the progressive nature of these diseases.
Alarmins S100A8/A9 promote intervertebral disc degeneration and inflammation-related pain in a rat model through toll-like receptor-4 and activation of the NF-κB signaling pathway.
Osteoarthritis and cartilage
OBJECTIVE:The molecules released from cells undergoing necrosis are recognized as alarmins, and S100A8/9, a typical alarmin, is associated with several inflammation-related diseases. This study was to investigate the molecular role of S100A8/A9 on the process of intervertebral disc degeneration (IVDD) and inflammation-related pain. METHODS:The expression pattern of S100A8/A9 in different degenerated human nucleus pulposus (NP) tissues were measured by Real-time quantitative reverse transcription PCR (RT-qPCR) and immunohistochemical (IHC). The effects of S100A8/A9 on matrix production were assessed by RT-qPCR, western blotting, and cell immunofluorescence. Involvement of TLR4 and NF-κB signaling pathways were studied by pharmachemical inhibitors and small interfering RNAs (siRNAs). The development of degenerative and pain features in the IVDD model were examed by IHC and pain-behavior testing. RESULTS:The expression of S100A8/A9 was significantly elevated in severely degenerated human NP tissue with similar expression pattern of TNF-α. In NP cells, S100A8/A9 increased MMP-3/13, TNF-α, IL-6 expression and inhibited aggrecan and collagen II expression. RT-qPCR and western blotting showed that the regulatory effects of S100A8/A9 on IVD were TLR4 dependent. Pharmacological inhibition or siRNA knockdown of the NF-κB signaling attenuated S100A8/A9-induced upregulation of MMP-3/13, TNF-α and IL-6. In vivo, S100A9 inhibitor treatment inhibited disc-puncture induced IVDD and inflammation-related pain. CONCLUSIONS:This study showed that S100A8/A9 bound to TLR4 and increased the expression of MMPs, TNF-α, and IL-6 through NF-κB signaling pathways in NP cells. Furthermore, S100A8/A9 inhibitor could prevent development of IVDD and inflammation-related pain in the rat model.
10.1016/j.joca.2022.03.011
Karacoline, identified by network pharmacology, reduces degradation of the extracellular matrix in intervertebral disc degeneration via the NF-κB signaling pathway.
Zhou Xiaoli,Hong Yingying,Zhan Yulin
Journal of pharmaceutical analysis
Karacoline is a compound found in the plant Reichb. Although Reichb is widely used for the treatment of pain, very few studies have been carried out on the use of karacoline due to its potential toxicity. In this study, we selected key matrix metalloproteinases (MMPs), collagen II, and aggrecan as targets due to their association with intervertebral disc degeneration (IDD). Using these targets, we then used network pharmacology to predict a series of molecules that might exert therapeutic effects on IDD. Of these molecules, karacoline was predicted to have the best effect. Tumor necrosis factor (TNF)-α is known to promote the degeneration of the extracellular matrix in IDD. We therefore applied different concentrations of karacoline (0, 1.25, or 12.88 μM) along with 100 ng/mL TNF-α to rat nucleus pulposus cells and found that karacoline reduced the expression of MMP-14 in IDD by inhibiting the nuclear factor (NF)-κB pathway, while collagen II and aggrecan expression was increased. This suggested that extracellular matrix degradation was inhibited by karacoline ( < 0.05). Our data therefore reveal a new clinical application of karacoline and provide support for the use of network pharmacology in predicting novel drugs.
10.1016/j.jpha.2019.07.002
Time course investigation of intervertebral disc degeneration in a rat-tail puncture model.
Chen Chia-Hsian,Chiang Chang-Jung,Wu Lien-Chen,Yang Chih-Hong,Kuo Yi-Jie,Tsuang Yang-Hwei,Tsai Tung-Hu
Life sciences
AIMS:Intervertebral disc (IVD) degeneration was believed to contribute to lower back pain. The aim of the study was to investigate the pathogenesis and regulatory mechanism of puncture-induced IVD degeneration. MAIN METHODS:We established a rat-tail puncture model using Kirschner wire and a homemade stopper. The progress of disc degeneration was evaluated by histological examination and the quantitative measurement of type I, type II collagen and other factors expression at 0.5, 1, 2, 6, and 12weeks after puncture and was compared with control rats of the same age. KEY FINDINGS:Histological examination and Safranin-O staining revealed progressive degeneration of the punctured disc. Matrix metalloproteinase 13 (MMP13) was increased at 1week after puncture but did not change in the control group. The interleukin-1 beta (IL-1β) mRNA expression level was elevated at the acute stage after puncture compared with the control group. The hypoxia inducible factor 2 (HIF-2) increased expression in punctured groups. Additionally, compare to adjacent non-punctured segments, HIF-2α expression level transiently increased and then decreased in the nucleus pulposus immediately following puncture, and it then increased 12weeks after puncture. SIGNIFICANCE:The degenerative changes observed in this rat-tail puncture model are similar to human disc degeneration and that this model may be valuable for elucidating the molecular mechanisms and pathways underlying disc degeneration.
10.1016/j.lfs.2016.05.020
17β-Estradiol alleviates intervertebral disc degeneration by inhibiting NF-κB signal pathway.
Life sciences
AIM:To investigate the effect of 17β-Estradiol (E2) on intervertebral disc degeneration (IVDD) and the related mechanism. MATERIALS AND METHODS:Immunohistochemistry was used to detect the expression of estrogen receptor β (ERβ) within intervertebral discs of humans and rats. After that, rat IVDD model was established by needle puncture and bilateral ovariectomy. Then, the serum E2 level was detected by enzyme linked immunosorbent assay, and the degree of IVDD was evaluated by X-ray, magnetic resonance imaging, hematoxylin and eosin staining, and Safranin O-Fast Green staining. Finally, we used immunohistochemistry and immunofluorescence staining to determine the effect of E2 on nuclear factor kappa-B (NF-κB) signal pathway both in vivo and in vitro. KEY FINDINGS:We identified that IVDD was associated with lower levels of ERβ and ERβ levels were inversely correlated with IVDD. The histological staining and radiological results showed that E2 supplement could alleviate IVDD progression. Additionally, immunohistochemistry staining demonstrated that E2 could inhibit nucleus pulposus cell (NPC) apoptosis, matrix metalloproteinases (MMPs) synthesis, and degradation of extracellular matrix (ECM) by inhibiting the activation of NF-κB signal pathway. Furthermore, immunofluorescence staining showed that the above effects of E2 on the NF-κB signal pathway could be blocked by the estrogen receptor antagonist ICI182780 in vitro. Finally, inhibition of NF-κB signal pathway by BAY11-7082 could reduce MMPs synthesis and ECM degradation of NPCs. SIGNIFICANCE:Collectively, these findings indicated that E2 could effectively ameliorate IVDD by inhibiting NPC apoptosis via inhibition of NF-κB signal pathway.
10.1016/j.lfs.2021.119874
Circular RNA derived from TIMP2 functions as a competitive endogenous RNA and regulates intervertebral disc degeneration by targeting miR‑185‑5p and matrix metalloproteinase 2.
Guo Wei,Zhang Bin,Sun Chao,Duan Hui-Quan,Liu Wei-Xiao,Mu Kun,Zhao Ling,Li Hao-Ran,Dong Zhan-Yin,Cui Qing
International journal of molecular medicine
Intervertebral disc degeneration (IDD) is an important cause of lower back pain, although the underlying mechanisms remain poorly understood. The present study aimed to examine the role of a circular RNA derived from tissue inhibitor of metallopeptidases 2 (circ‑TIMP2) in degenerative nucleus pulposus (NP) tissues, and to validate its function in cultured human NP cells. Overexpression of miR‑185‑5p in NP cells markedly inhibited the enhanced extracellular matrix (ECM) catabolism induced by tumor necrosis factor‑α (TNF‑α) and interleukin‑1β (IL‑1β) treatment. Bioinformatics analysis demonstrated that matrix metalloproteinase 2 (MMP2) was a potential target of miR‑185‑5p. MMP2 protein expression levels were increased following treatment with TNF‑α and IL‑1β in NP cells compared with those in untreated cells, and this effect was attenuated by transfection with miR‑185‑5p. Compared with normal NP tissues, IDD samples exhibited higher circ‑TIMP2 expression levels. In addition, overexpression of circ‑TIMP2 promoted ECM catabolism and suppressed ECM anabolism. Furthermore, circ‑TIMP2 sequestered miR‑185‑5p, which may potentially upregulate the target genes associated with ECM degradation. In conclusion, the results of the present study revealed that circ‑TIMP2 promoted TNF‑α‑ and IL‑1β‑induced NP cell imbalance between ECM anabolism and catabolism via miR‑185‑5p‑MMP2 signaling. These findings provide a potential therapeutic option for the treatment of IDD.
10.3892/ijmm.2020.4621
LINC01121 induced intervertebral disc degeneration via modulating miR-150-5p/MMP16 axis.
Chen Xin,Li Zheng,Xu Derong,Li Shugang
The journal of gene medicine
BACKGROUND:Growing evidence indicates that Long noncoding RNAs contribute to cell differentiation, invasion, metabolism, proliferation and metastasis. However, the potential role of LINC01121 in progression of intervertebral disc degeneration (IDD) remains unclear. METHODS:LINC01121, matrix metalloprotease (MMP)-16 and miR-150-5p expression was determined by a quantitative-reverse transcriptase-polymerase chain reaction assay. Inflammatory cytokines level was measured by an enzyme-linked immunosorbent assay and cell counting kit-8 analysis was used to assess cell proliferation. MMP-16-specific binding with miR-150-5p was verified with a luciferase reporter assay. RESULTS:We noted that interleukin (IL)-1β and tumor necrosis factor (TNF)-α treatment enhanced LINC01121 and MMP-16 expression in nucleus pulposus (NP) cells. LINC01121 was higher in IDD specimens compared to that in control specimens. Higher expression of LINC01121 was correlated with disc degeneration degree. Ectopic expression of LINC01121 enhanced cell proliferation and promoted ki-67, MMP-3 and ADAMTS5 expression and also suppressed collagen II expression in NP cells. We observed that overexpression of LINC01121 increased the secretion of three inflammatory cytokines, including IL-6, TNF-α and IL-1β. We found that ectopic expression of LINC01121 decreased the miR-150-5p level in NP cells. Luciferase reporter data confirmed that MMP-16 was one direct target of miR-150-5p. Overexpression of miR-150-5p inhibited MMP-16 level and elevated the expression of LINC01121 enhanced MMP-16 level. We also found that MMP-16 was up-regulated in IDD specimens compared to that in control specimens. Higher expression of MMP-16 was correlated with disc degeneration degree. Interestingly, MMP-16 expression was positively related to LINC01121 in IDD specimens. Finally, overexpression of LINC01121 regulated cell growth, extracellular matrix degradation and inflammatory cytokine secretion via modulating MMP-16. CONCLUSIONS:our data suggested LINC01121 may be a new therapeutic target for IDD.
10.1002/jgm.3231
Matrix metalloproteinase expression levels suggest distinct enzyme roles during lumbar disc herniation and degeneration.
Bachmeier Beatrice E,Nerlich Andreas,Mittermaier Norbert,Weiler Christoph,Lumenta Christianto,Wuertz Karin,Boos Norbert
European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society
The disruption of the extracellular disc matrix is a major hallmark of disc degeneration. This has previously been shown to be associated with an up-regulation of major matrix metalloproteinase (MMP) expression and activity. However, until now hardly any data are available for MMP/TIMP regulation and thereby no concept exists as to which MMP/TIMP plays a major role in disc degeneration. The objective of this study was, therefore, to identify and quantify the putative up-regulation of MMPs/TIMPs on the mRNA and protein level and their activity in disc material in relation to clinical data and histological evidence for disc degeneration. A quantitative molecular analysis of the mRNA expression levels for the MMPs (MMPs-1, -2, -3, -7, -8, -9, -13) and the MMP inhibitors (TIMPs-1 and -2) was performed on 37 disc specimens obtained from symptomatic disc herniation or degeneration. In addition, disc specimens from patients without disc degeneration/herniation (=controls) were analyzed. Expression of MMPs-1, -2, -3, -7, -8, -9, -13 and TIMPs-1, -2 was analyzed using quantitative RT-PCR, normalized to the expression level of a house keeping gene (GAPDH). Gene expression patterns were correlated with MMP activity (in situ zymography), protein expression patterns (immunohistochemistry), degeneration score (routine histology) and clinical data. MMP-3 mRNA levels were consistently and substantially up-regulated in samples with histological evidence for disc degeneration. A similar but less pronounced up-regulation was observed for MMP-8. This up-regulation was paralleled by the expression of TIMP-1 and to a lesser extent TIMP-2. In general, these findings could be confirmed with regard to protein expression and enzyme activity. This study provides data on the gene and protein level, which highlights the key role of MMP-3 in the degenerative cascade leading to symptomatic disc degeneration and herniation. Control of the proteolytic activity of MMP-3 may, therefore, come into the focus when aiming to develop new treatment options for early disc degeneration.
10.1007/s00586-009-1031-8
Cellular senescence - Molecular mechanisms of intervertebral disc degeneration from an immune perspective.
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Intervertebral disc degeneration (IVDD) is a frequent and intractable chronic condition in orthopedics that causes enormous discomfort in patients' lives and thoughts, as well as a significant economic burden on society and the nation. As a result, understanding the pathophysiology of IVDD is critical. The pathophysiology of IVDD has been linked to numerous variables, including oxidative stress, apoptosis, matrix metalloproteinases, and inflammatory factors. Cellular senescence has recently attracted a lot of attention in the study of age-related diseases. It has been discovered that IVDD is intimately linked to human senescence, in which nucleus pulposus cell senescence may play a significant role. Previously, our group did a comprehensive and systematic clarification of the pathogenesis of IVDD from an immune perspective and discovered that the fundamental pathogenesis of IVDD is inflammatory upregulation and nucleus pulposus cell death caused by an imbalance in the immune microenvironment. In this review, we will treat nucleus pulposus cell senescence as a novelty point to clarify the pathophysiology of IVDD and further explore the probable relationship between senescence and immunity along with the dysregulation of the immunological microenvironment to propose new therapeutic approaches for IVDD.
10.1016/j.biopha.2023.114711
Dysregulated miR-127-5p contributes to type II collagen degradation by targeting matrix metalloproteinase-13 in human intervertebral disc degeneration.
Hua Wen-Bin,Wu Xing-Huo,Zhang Yu-Kun,Song Yu,Tu Ji,Kang Liang,Zhao Kang-Cheng,Li Shuai,Wang Kun,Liu Wei,Shao Zeng-Wu,Yang Shu-Hua,Yang Cao
Biochimie
BACKGROUND:Intervertebral disc degeneration (IDD) is a chronic disease associated with the degradation of extracellular matrix (ECM). Matrix metalloproteinase (MMP)-13 is a major enzyme that mediates the degradation of ECM components. MMP-13 has been predicted to be a potential target of miR-127-5p. However, the exact function of miR-127-5p in IDD is still unclear. OBJECTIVE:We designed this study to evaluate the correlation between miR-127-5p level and the degeneration of human intervertebral discs and explore the potential mechanisms. METHODS:miR-127-5p levels and MMP-13 mRNA levels were detected by quantitative real-time polymerase chain reaction (qPCR). To determine whether MMP-13 is a target of miR-127-5p, dual luciferase reporter assays were performed. miR-127-5p mimic and miR-127-5p inhibitor were used to overexpress or downregulate miR-127-5p expression in human NP cells, respectively. Small interfering RNA (siRNA) was used to knock down MMP-13 expression in human NP cells. Type II collagen expression in human NP cells was detected by qPCR, western blotting, and immunofluorescence staining. RESULTS:We confirmed that miR-127-5p was significantly downregulated in nucleus pulposus (NP) tissue of degenerative discs and its expression was inversely correlated with MMP-13 mRNA levels. We reveal that MMP-13 may act as a target of miR-127-5p. Expression of miR-127-5p was inversely correlated with type II collagen expression in human NP cells. Moreover, suppression of MMP-13 expression by siRNA blocked downstream signaling and increased type II collagen expression. CONCLUSION:Dysregulated miR-127-5p contributed to the degradation of type II collagen by targeting MMP-13 in human IDD. Our findings highlight that miR-127-5p may serve as a new therapeutic target in IDD.
10.1016/j.biochi.2017.05.018
The role of matrix metalloproteinase 9 in intervertebral disc degeneration.
Zigouris Andreas,Alexiou George A,Batistatou Anna,Voulgaris Spyridon,Kyritsis Athanasios P
Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia
The present study investigates the histological alterations and expression of matrix metalloproteinase 9 (MMP-9) in disc specimens of 43 patients who underwent surgery for lumbar disc herniation. The immunostaining for MMP-9 was evaluated semi-quantitatively. Histologic degeneration was scored between 0 and 12 depending on the degree of chondrocyte proliferation and presence of tears and clefts, granular changes and mucous degeneration. Herniation was graded as grade 1 (protrusion), grade 2 (extrusion) or grade 3 (sequestration) on MRI. Although there was no significant statistical difference between the histologic degeneration score and age, degenerative changes were more pronounced in higher grade of herniation (p<0.0001). MMP-9 expression was related to histologic degenerative score in all age groups (p=0.0065). MMP-9 was also related to herniation grade in patients younger than 30 years of age (p=0.0037). No significant association was found between MMP-9 expression and herniation grade in patients who were 30-60 years or over 60 years of age.
10.1016/j.jocn.2011.01.036
Pathogenesis and therapeutic implications of matrix metalloproteinases in intervertebral disc degeneration: A comprehensive review.
Biochimie
Intervertebral disc (IVD) degeneration (IDD) is a common disorder that affects the spine and is a major cause of lower back pain (LBP). The extracellular matrix (ECM) is the structural foundation of the biomechanical properties of IVD, and its degradation is the main pathological characteristic of IDD. Matrix metalloproteinases (MMPs) are a group of endopeptidases that play an important role in the degradation and remodeling of the ECM. Several recent studies have shown that the expression and activity of many MMP subgroups are significantly upregulated in degenerated IVD tissue. This upregulation of MMPs results in an imbalance of ECM anabolism and catabolism, leading to the degradation of the ECM and the development of IDD. Therefore, the regulation of MMP expression is a potential therapeutic target for the treatment of IDD. Recent research has focused on identifying the mechanisms by which MMPs cause ECM degradation and promote IDD, as well as on developing therapies that target MMPs. In summary, MMP dysregulation is a crucial factor in the development of IDD, and a deeper understanding of the mechanisms involved is needed to develop effective biological therapies that target MMPs to treat IDD.
10.1016/j.biochi.2023.05.015
Suramin attenuates intervertebral disc degeneration by inhibiting NF-κB signalling pathway.
Liu Zi-Miao,Lu Cheng-Chang,Shen Po-Chih,Chou Shih-Hsiang,Shih Chia-Lung,Chen Jian-Chih,Tien Yin Chun
Bone & joint research
AIMS:Interleukin (IL)-1β is one of the major pathogenic regulators during the pathological development of intervertebral disc degeneration (IDD). However, effective treatment options for IDD are limited. Suramin is used to treat African sleeping sickness. This study aimed to investigate the pharmacological effects of suramin on mitigating IDD and to characterize the underlying mechanism. METHODS:Porcine nucleus pulposus (NP) cells were treated with vehicle, 10 ng/ml IL-1β, 10 μM suramin, or 10 μM suramin plus IL-1β. The expression levels of catabolic and anabolic proteins, proinflammatory cytokines, mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB-related signalling molecules were assessed by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence analysis. Flow cytometry was applied to detect apoptotic cells. The ex vivo effects of suramin were examined using IDD organ culture and differentiation was analyzed by Safranin O-Fast green and Alcian blue staining. RESULTS:Suramin inhibited IL-1β-induced apoptosis, downregulated matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5, and upregulated collagen 2A (Col2a1) and aggrecan in IL-1β-treated NP cells. IL-1β-induced inflammation, assessed by IL-1β, IL-8, and tumour necrosis factor α (TNF-α) upregulation, was alleviated by suramin treatment. Suramin suppressed IL-1β-mediated proteoglycan depletion and the induction of MMP-3, ADAMTS-4, and pro-inflammatory gene expression in ex vivo experiments. CONCLUSION:Suramin administration represents a novel and effectively therapeutic approach, which could potentially alleviate IDD by reducing extracellular matrix (ECM) deposition and inhibiting apoptosis and inflammatory responses in the NP cells. Cite this article: 2021;10(8):498-513.
10.1302/2046-3758.108.BJR-2020-0041.R3
MMPs and ADAMTSs in intervertebral disc degeneration.
Wang Wen-Jun,Yu Xiao-Hua,Wang Cheng,Yang Wei,He Wen-Si,Zhang Shu-Jun,Yan Yi-Guo,Zhang Jian
Clinica chimica acta; international journal of clinical chemistry
Intervertebral disc degeneration (IDD) is the most common diagnosis in patients with low back pain, a leading cause of musculoskeletal disability worldwide. The major components of extracellular matrix (ECM) within the discs are type II collagen (Col II) and aggrecan. Excessive destruction of ECM, especially loss of Col II and aggrecan, plays a critical role in promoting the occurrence and development of IDD. Matrix metalloproteinases (MMPs) and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTSs) are primary enzymes that degrade collagens and aggrecan. There is a large and growing body of evidence that many members of MMPs and ADAMTSs are highly expressed in degenerative IVD tissue and cells, and are closely involved in ECM breakdown and the process of disc degeneration. In contrast, targeting these enzymes has shown promise for promoting ECM repair and mitigating disc regeneration. In the current review, after a brief description regarding the biology of MMPs and ADAMTSs, we mainly focus on their expression profiles, roles and therapeutic potential in IDD. A greater understanding of the catabolic pathways involved in IDD will help to develop potential prophylactic or regenerative biological treatment for degenerative disc disease in the future.
10.1016/j.cca.2015.06.023
Evaluation of the Association Between Matrix Metalloproteinase 11 and Intervertebral Disc Disease.
Aras Adem Bozkurt,Guven Mustafa,Balak Naci,Ayan Erdoğan,Uyar Suheyla Bozkurt,Elmaci Ilhan
Turkish neurosurgery
AIM:The intervertebral disc starts to degenerate when a human being begins to stand and learn to walk. It is known that many extrinsic, intrinsic and genetic factors play a role in disc degeneration. In this study, we examined whether the matrix metalloproteinase 11 might be associated with intervertebral disc degeneration. MATERIAL AND METHODS:Fifty-six patients with lumbar disc herniations who were operated at Göztepe Education and Research Hospital, Neurosurgery Clinic between September 2008 and December 2009 were prospectively reviewed. History and complaints were obtained from the case reports. Neuroradiological evaluation was performed with magnetic resonance imaging. Surgical findings of cases were reported in the operation notes. Microscopic posterior hemipartial laminectomy and discectomy were performed in all cases. Degenerated herniated disc material of all cases extracted during surgery was evaluated with immunohistochemical staining in Marmara University, Institute of Neurological Sciences, Pathology Laboratory. RESULTS:Comparing the immunohistochemical staining of cases who were 50 years or younger and cases who were over 50 years old, statistical significance was determined. CONCLUSION:Matrix metalloproteinase 11 has a role in degenerating intervertebral disc disease, but it is not the only factor. Matrix metalloproteinase 11 might be a genetic factor in young-middle aged patients.
10.5137/1019-5149.JTN.12762-14.0
Exogenous Klotho ameliorates extracellular matrix degradation and angiogenesis in intervertebral disc degeneration via inhibition of the Rac1/PAK1/MMP-2 signaling axis.
Mechanisms of ageing and development
Intervertebral disc degeneration (IDD) is highly ubiquitous in the aged population and is an essential factor for low back pain and spinal disability. Because of the association between IDD and senescence, we investigated the ability of the anti-aging drug Klotho to inhibit age-dependent advancement of nucleus pulposus cell (NPC) degeneration. The results indicated that 400 pM exogenous Klotho significantly ameliorated extracellular matrix degradation and angiogenesis. Moreover, we demonstrated that the suppression of angiogenesis and extracellular matrix catabolism was related to inhibition of the Ras-related C3 botulinum toxin substrate 1 (Rac1)/PAK1 axis and matrix metalloproteinase 2 protein expression by exogenous Klotho cotreatment with a Rac1 inhibitor, gene overexpression in NPCs, and stimulation of human umbilical vein endothelial cells with conditioned medium from NPCs. The treatment also preserved the NPC phenotype, viability, and matrix content. In conclusion, these results suggest that the new anti-aging drug Klotho is a potential treatment strategy to mitigate IDD, and thus, provides an innovative understanding of the molecular mechanism of IDD. DATA AVAILABILITY: All data supporting the findings of this study are available from the corresponding authors upon reasonable request.
10.1016/j.mad.2022.111715
Immuohistochemical score of matrix metalloproteinase-1 may indicate the severity of symptomatic cervical and lumbar disc degeneration.
Hsu Hsien-Ta,Yue Chung-Tai,Teng Ming-Sheng,Tzeng I-Shiang,Li Tin-Chou,Tai Po-An,Huang Kuo-Feng,Chen Cheng-Yu,Ko Yu-Lin
The spine journal : official journal of the North American Spine Society
BACKGROUND CONTEXT:Intervertebral disc (IVD) degeneration is related to numerous risk factors, including obesity. Leptin, one of the commonly measured adipokines, is proven to play an important role in the pathogenesis of IVD degeneration. In the context of IVD degeneration, matrix metalloproteinase-1 (MMP-1), which is upregulated and activated by leptin, is the most abundant catabolic enzyme. It remains unclear which of the factors mentioned above is most strongly associated with IVD degeneration. PURPOSE:To investigate the influence of MMP-1 in IVD degeneration, we determined the strength of different predictors, including age, sex, magnetic resonance imaging (MRI), Modic changes (MCs), body mass index (BMI), leptin, and MMP-1. This was achieved by assessing the correlation among these factors and histologic degeneration score (HDS). STUDY DESIGN:This study included 89 patients undergoing cervical discectomy for disc herniation, 93 who underwent lumbar discectomy, and 90 control subjects. Herniated disc tissue and plasma were used after the study was approved by the Human Ethics Review Committee at the authors' institution. METHODS:Hematoxylin and eosin (H&E), Alcian blue-PAS and immunohistochemical (IHC) staining were performed to measure the expression levels of leptin and MMP-1. Circulating plasma levels of leptin and MMP-1 were measured using an enzyme-linked immunosorbent assay. To assess the correlation with HDS, measurements of age, sex, BMI, MRI scale, MCs scale, leptin/MMP-1 plasma concentration, and leptin/MMP-1 IHC expression were analyzed. RESULTS:Patients with cervical or lumbar discectomy had significantly higher BMI than controls. Significantly more men than women were involved in the lumbar patients as compared with the cervical patients and the control subjects. After adjustment for age and sex, plasma leptin and leptin IHC score correlated significantly with BMI in patients with cervical or lumbar discectomy. Age, sex, MRI scale, MCs scale, and leptin/MMP-1 plasma concentration were not positively correlated with HDS. HDS was significantly associated with BMI, leptin IHC score, and MMP-1 IHC score. After a stepwise-multiple linear regression analysis to evaluate the strength of the correlations between HDS and various factors, only the MMP-1 IHC score demonstrated an independent association with HDS in patients with degeneration of the cervical or lumbar disc. CONCLUSIONS:MMP-1 IHC score is an independent predictor of the severity of cervical or lumbar IVD degeneration. CLINICAL SIGNIFICANCE:MMP-1 IHC score may be used as an indicator of IVD degeneration.
10.1016/j.spinee.2019.08.004
Current Knowledge and Future Therapeutic Prospects in Symptomatic Intervertebral Disc Degeneration.
Kim Joo Han,Ham Chang Hwa,Kwon Woo-Keun
Yonsei medical journal
Intervertebral disc (IVD) degeneration is the main source of intractable lower back pain, and symptomatic IVD degeneration could be due to different degeneration mechanisms. In this article, we describe the molecular basis of symptomatic IVD degenerative disc diseases (DDDs), emphasizing the role of degeneration, inflammation, angiogenesis, and extracellular matrix (ECM) regulation during this process. In symptomatic DDD, pro-inflammatory mediators modulate catabolic reactions, resulting in changes in ECM homeostasis and, finally, neural/vascular ingrowth-related chronic intractable discogenic pain. In ECM homeostasis, anabolic protein-regulating genes show reduced expression and changes in ECM production, while matrix metalloproteinase gene expression increases and results in aggressive ECM degradation. The resultant loss of normal IVD viscoelasticity and a concomitant change in ECM composition are key mechanisms in DDDs. During inflammation, a macrophage-related cascade is represented by the secretion of high levels of pro-inflammatory cytokines, which induce inflammation. Aberrant angiogenesis is considered a key initiative pathologic step in symptomatic DDD. In reflection of angiogenesis, vascular endothelial growth factor expression is regulated by hypoxia-inducible factor-1 in the hypoxic conditions of IVDs. Furthermore, IVD cells undergoing degeneration potentially enhance neovascularization by secreting large amounts of angiogenic cytokines, which penetrate the IVD from the outer annulus fibrosus, extending deep into the outer part of the nucleus pulposus. Based on current knowledge, a multi-disciplinary approach is needed in all aspects of spinal research, starting from basic research to clinical applications, as this will provide information regarding treatments for DDDs and discogenic pain.
10.3349/ymj.2022.63.3.199
Genetic aspects of intervertebral disc degeneration.
Hanaei Sara,Abdollahzade Sina,Khoshnevisan Alireza,Kepler Christopher K,Rezaei Nima
Reviews in the neurosciences
Intervertebral disc degeneration (IVDD) is one of the common causes of low back pain. Similar to many other multifactorial diseases, it is affected by environmental and genetic factors. Although not completely understood, genetic factors include a wide spectrum of variations, such as single nucleotide polymorphisms, which could play a significant role in the etiology of this disease. Besides, the interactions with environmental factors could make the role of genetic factors more complicated. Genetic variations in disc components could participate in developing degenerative disc disease through altering the normal homeostasis of discs. Gene polymorphisms in disc proteins (collagens I, II, III, IX, and XI), proteoglycans (aggrecan), cytokines (interleukins I, VI, and X), enzymes (matrix metalloproteinases II, III, and IX), and vitamin D receptor seem to play considerable roles in the pathology of this disease. There are also many other investigated genes that could somehow take part in the process. However, it seems that more studies are needed to clarify the exact role of genetics in IVDD.
10.1515/revneuro-2014-0077
Matrix metalloproteinases: the clue to intervertebral disc degeneration?
Goupille P,Jayson M I,Valat J P,Freemont A J
Spine
STUDY DESIGN:A review of the current literature on the role of matrix metalloproteinases in intervertebral disc degeneration. OBJECTIVE:To detail the characteristics of matrix metalloproteinases (classification, structure, substrate specificity and regulation) and to report previous studies of intervertebral discs. SUMMARY OF BACKGROUND DATA:Degeneration of the intervertebral disc, a probable prerequisite to disc herniation, is a complex phenomenon, and its physiopathologic course remains unclear. Matrix metalloproteinases probably play an important role but have received sparse attention in the literature. METHODS:A systematic review of studies reporting a role of matrix metalloproteinases in intervertebral disc degeneration. RESULTS:In several studies, investigators have reported the presence of proteolytic enzymes from disc culture systems and disc tissue extracts in degenerated human intervertebral discs, especially collagenase-1 (MMP-1) and stromelysin-1 (MMP-3). The matrix metalloproteinases are regulated by specific inhibitors (tissue inhibitors of metalloproteinases, or TIMPS), cytokines (interleukin-1), and growth factors. CONCLUSIONS:This field of application is of particular interest because conventional treatments are disappointing in chronic low back pain. Clinical trials with specific inhibitors of metalloproteinases are beginning in osteoarthritis.
Bioactive lipids in intervertebral disc degeneration and its therapeutic implications.
Bioscience reports
Intervertebral disc (IVD) degeneration is not uncommon. It is estimated that approximately >60% of individuals above the age of 40 years suffer from IVD degeneration. Shan et al. showed that hyperglycemia can enhance apoptosis of anulus fibrosis cells in a JNK pathway and p38 mitogen-activated protein kinase (MAPK) pathway dependent fashion. Recent studies showed that IVD degeneration could be an inflammatory condition characterized by increased production of matrix metalloproteinases, TNF-α, nitric oxide, IL-6, IL-17, IL-9, and prostaglandin E2, and decreased formation of anti-inflammatory molecules such as lipoxin A4. This imbalance between pro- and anti-inflammatory molecules seem to activate JNK pathway and p38 MAPK pathway to induce apoptosis of anulus fibrosis and nucleus pulposus cells. The activation of production of PGE2 (due to activation of COX-2 pathway) seems to be dependent on p38/c-Fos and JNK/c-Jun activation in an AP-1-dependent manner. These results imply that suppressing pro-inflammatory events in the disc by either augmenting anti-inflammatory events or suppressing production of pro-inflammatory molecules or both may form a logical step in the prevention and management of IVD degeneration.
10.1042/BSR20192117
Mitochondrial Dysfunction in Oxidative Stress-Mediated Intervertebral Disc Degeneration.
Orthopaedic surgery
Intervertebral disc degeneration (IVDD) is the most common contributor to low back pain (LBP). Recent studies have found that oxidative stress and reactive oxygen species (ROS) play an important role in IVDD. As a by-product of aerobic respiration, ROS is mainly produced in the mitochondria by the electron transport chain and other mitochondrial located proteins. With the excessive accumulation of ROS, mitochondria are also the primary target of ROS attack in disc cells. A disrupted balance between intracellular ROS production and antioxidant capacity will lead to oxidative stress, which is the key contributor to cell apoptosis, cell senescence, excessive autophagy, and mitochondrial dysfunction. As the pivotal ingredient of oxidative stress, mitochondrial dysfunction manifests as imbalanced mitochondrial dynamics and dysregulated mitophagy. Mitochondria can alter their own dynamics through the process of fusion and fission, so that disabled mitochondria can be separated from the mitochondrial pool. Moreover, mitophagy participates by clearing these dysfunctional mitochondria. Abnormality in any of these processes either increases the production or decreases the clearance of ROS, leading to a vicious cycle that results in the death of intervertebral disc cells in large quantities, combined with degradation of the extracellular matrix and overproduction of matrix metalloproteinase. In this review, we explain the changes in mitochondrial morphology and function during oxidative stress-mediated IVDD and highlight the important role of mitochondria in this process. Eventually, we summarize the IVDD therapeutic strategies targeting mitochondrial dysfunction based on current understanding of the role of oxidative stress in IVDD.
10.1111/os.13302
Genetic Dimension of Intervertebral Disc Degeneration: Polymorphism of Matrix Metalloproteinase 1 and 3 in the North Iranian Population.
Saberi Alia,Salehi Zivar,Naderinabi Bahram,Ansari Sahar Hojaat,Mashayekhi Sohail
Turkish neurosurgery
AIM:The etiology of intervertebral disc degeneration is still vague and both genetic and environmental factors are assumed as the main causes. One of the proposed genetic factors is the polymorphism of matrix metalloproteinases (MMPs) genes. The aim of this study was to explore the relationship between two polymorphisms (MMP-1-755 T/G [rs498186] and MMP-3 A/C [rs632478]) and disc degeneration. MATERIAL AND METHODS:We performed a case-control study on 130 cases with intervertebral disc degeneration confirmed by magnetic resonance imaging (MRI) and 210 healthy individuals. The Schneiderman criterion was used to determine the severity of the disc degeneration. Blood samples were collected from the participants. The genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Odds ratios and 95% CIs were determined as measures of the strength of association between genotypes and disc degeneration. RESULTS:The most frequent intervertebral disc degeneration was observed in age range of 31-40 years (43.2%). A significant association was found between the MMP-3 polymorphism and disc degeneration (p < 0.001). The homozygote CC was associated with an increased risk of disc degeneration compared with the AA genotype (OR=5.25; 95%CI=2.82-9.77, p < 0.001). We did not find any significant association of the MMP-1 polymorphism with disc degeneration (p=0.95). CONCLUSION:The MMP-3 [rs632478] polymorphism may contribute to susceptibility to disc degeneration. To confirm our findings, additional well-designed studies in diverse ethnic populations are required.
10.5137/1019-5149.JTN.19978-17.0
Intervertebral disc ageing and degeneration: The antiapoptotic effect of oestrogen.
Yang Sidong,Zhang Feng,Ma Jiangtao,Ding Wenyuan
Ageing research reviews
As an important part of the spinal column, the intervertebral disc (IVD) plays an important role in the intervertebral juncture and spinal movement in general. IVD degeneration (IVDD), which mimics disc ageing but at an accelerated rate, is a common and chronic process that results in severe spinal symptoms, such as lower back pain. It is generally assumed that lower back pain caused by IVDD can also develop secondary conditions, including spinal canal stenosis, spinal segmental instability, osteophyte formation, disc herniation and spinal cord and nerve root compression. Over the past few years, many researchers around the world have widely studied the relevance between oestrogen and IVDD, indicating that oestrogen can effectively alleviate IVDD development by inhibiting the apoptosis of IVD cells. Oestrogen can decrease IVD cell apoptosis in multiple ways, including the inhibition of the inflammatory cytokines IL-1β and TNF-α, reducing catabolism because of inhibition of matrix metalloproteinases, upregulating integrin αβ and IVD anabolism, activating the PI3K/Akt pathway, decreasing oxidative damage and promoting autophagy. In this article, we perform an overview of the literature regarding the antiapoptotic effect of oestrogen in IVDD.
10.1016/j.arr.2019.100978
Matrix metalloproteinase 12 is an indicator of intervertebral disc degeneration co-expressed with fibrotic markers.
Lv F-J,Peng Y,Lim F L,Sun Y,Lv M,Zhou L,Wang H,Zheng Z,Cheung K M C,Leung V Y L
Osteoarthritis and cartilage
OBJECTIVE:Recent evidence suggests a role of fibrogenesis in intervertebral disc (IVD) degeneration. We aim to explore if fibrotic genes may serve as IVD degeneration indicators, and if their expression is associated with myofibroblast activity. DESIGN:Transcriptional expression of fibrosis markers (COL1A1, COL3A1, FN1, HSP47, MMP12, RASAL1) were analyzed in degenerated (D) and non-degenerated (ND) human nucleus pulposus (NP) and annulus fibrosus (AF) cells, along with traditional (SOX9, ACAN) and newly established degeneration markers (CDH2, KRT19, KRT18, FBLN1, MGP, and COMP). Protein expression was investigated by immunohistochemistry in human IVDs, and in rodent IVDs undergoing natural ageing or puncture-induced degeneration. Co-expression with myofibroblast markers was examined by double staining on human and rat specimens. Disc degeneration severity and extent of fibrosis were determined by histological scoring and picrosirius red staining respectively. RESULTS:Human D-NP showed more intensive staining for picrosirius red than ND-NP. Among the genes examined, D-NP showed significantly higher MMP12 expression along with lower KRT19 expression. Protein expression analysis revealed increased MMP12(+) cells in human D-IVD. Histological scoring indicated mild degeneration in the punctured rat discs and discs of ageing mouse. Higher MMP12 positivity was found in peripheral NP and AF of the degenerative rat discs and in NP of the aged mice. In addition, human D-NP and D-AF showed increased α-SMA(+) cells, indicating enhanced myofibroblast activity. MMP12 was found co-expressed with α-SMA, FSP1 and FAP-α in human and rat degenerative IVDs. CONCLUSIONS:Our study suggests that in addition to a reduced KRT19 expression, an increased expression of MMP12, a profibrotic mediator, is characteristic of disc degenerative changes. Co-expression study indicates an association of the increased MMP12 positivity with myofibroblast activity in degenerated IVDs. Overall, our findings implicate an impact of MMP12 in disc cell homeostasis. The precise role of MMP12 in IVD degeneration warrants further investigation.
10.1016/j.joca.2016.05.012
Vitamin D Receptor Gene, Matrix Metalloproteinase 3 Polymorphisms and the Risk of Intervertebral Disc Degeneration Susceptibility: Meta-Analysis.
Nong Luming,Huang Yongjing,Zhao Shujie,Xu Nanwei
Asian spine journal
Several studies have evaluated the association between vitamin D receptor, matrix metalloproteinase 3 (MMP-3) polymorphisms and the risk of intervertebral disc degeneration susceptibility. The findings were inconsistent. This meta-analysis aimed to systematically assess the association between vitamin D receptor, MMP-3 polymorphisms and the risk of intervertebral disc degeneration susceptibility. A search of various databases was done covering all papers published until December 31th, 2014. Eight, 4, 3 studies were finally included that addressed the risk of intervertebral disc degeneration susceptibility and vitamin D receptor FokI (rs2228570), ApaI (rs7975232), and MMP-3 (rs731236) polymorphisms, respectively. FokI (f vs. F: summary odds ratio [OR], 1.13; 95% confidence interval [CI], 0.76-1.69; ff vs. FF: OR, 1.02; 95% CI, 0.59-1.77; ff vs. Ff/FF: OR, 1.05; 95% CI, 0.70-1.58), ApaI (a vs. A: OR, 0.73; 95% CI, 0.45-1.19; aa vs. AA: OR, 0.53; 95% CI, 0.22-1.25 =0.14; aa vs. AA/Aa: OR, 0.69; 95% CI, 0.53-0.89) in the vitamin D receptor gene and MMP3 polymorphisms (5A vs. 6A: OR, 1.92; 95% CI, 0.77-4.80; 5A5A vs. 6A6A: OR, 2.17; 95% CI, 0.75-6.24; 5A5A vs. 5A6A/6A6A: OR, 1.58; 95% CI, 0.72-3.44) were not obviously associated with risk of intervertebral disc degeneration susceptibility. FokI, ApaI polymorphisms in the vitamin D receptor gene and MMP-3 polymorphism are not obvious risk factors for intervertebral disc degeneration susceptibility.
10.4184/asj.2016.10.5.964
Characteristics and mechanisms of resorption in lumbar disc herniation.
Arthritis research & therapy
Lumbar disc herniation (LDH) can be spontaneously absorbed without surgical treatment. However, the pathogenesis and physiological indications for predicting protrusion reabsorption are still unclear, which prevents clinicians from preferentially choosing conservative treatment options for LDH patients with reabsorption effects. The purpose of this review was to summarize previous reports on LDH reabsorption and to discuss the clinical and imaging features that favor natural absorption. We highlighted the biological mechanisms involved in the phenomenon of LDH reabsorption, including macrophage infiltration, inflammatory responses, matrix remodeling, and neovascularization. In addition, we summarized and discussed potential clinical treatments for promoting reabsorption. Current evidence suggests that macrophage regulation of inflammatory mediators, matrix metalloproteinases, and specific cytokines in intervertebral disc is essential for the spontaneous reabsorption of LDH.
10.1186/s13075-022-02894-8
Melatonin modulates IL-1β-induced extracellular matrix remodeling in human nucleus pulposus cells and attenuates rat intervertebral disc degeneration and inflammation.
Zhang Yan,He Fan,Chen Zhi,Su Qihang,Yan Meijun,Zhang Qiang,Tan Jun,Qian Lie,Han Yingchao
Aging
The inflammatory-associated factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are widely reported to be associated with intervertebral disc (IVD) degeneration (IVDD). N-acetyl-5-methoxytryptamine (melatonin) is a natural hormone secreted by the pineal gland which has been shown to participate in several physiological and pathological progresses, such as aging, anti-inflammation, anti-apoptosis and autophagy regulation. However, the effects of melatonin on IVD remain unclear. In the present study, we treated human nucleus pulposus cells (NPCs) with melatonin and discovered that melatonin could modulate extracellular matrix (ECM) remodeling induced by IL-1β by enhancing collagen II and aggrecan expression levels and by downregulating matrix metalloproteinase-3 (MMP-3) levels. These findings were verified by western blot and immunofluorescence assays. Intraperitoneal injection of melatonin mitigated IVDD in the rat tail puncture model. X-ray and magnetic resonance imaging (MRI), as well as hematoxylin-eosin (H&E), Safranine O-Green, Alcian blue and Celium red staining methods were adopted to evaluate IVDD grades, the structural integrity of nucleus pulposus (NP) and annulus fibrosus (AF) and the damage and calcification of the cartilage endplate. Melatonin reduced inflammatory cell aggregation and the release of the inflammatory factors IL-1β, IL-6, TNF-α as determined by immunohistochemistry. In conclusion, the present study demonstrated that melatonin could modulate ECM remodeling by IL-1β in vitro and attenuate the IVDD and induction of inflammation in a rat tail puncture model in vivo. The data demonstrated that melatonin may contribute to the restoration processs of IVD following damage and may be used as a potential novel therapy for IVDD.
10.18632/aging.102472