Osteoarthritis (OA) is a prevalent degenerative disease that impairs limb function, and its pathogenesis is closely related to inflammation. Sakuranetin (SK) is a cherry flavonoid phytoalexin with potent anti-inflammatory, anti-oxidant, and ant-ifungal properties. In recent studies, flavonoid and phytoalexin-related medicines have shown promise in the treatment of OA. However, the effects of SK on chondrocyte inflammation and the chondrogenesis process have remained unexplored, as have its functions in OA treatment. This study sought to confirm the therapeutic effects of SK in the OA rat model and reveal the potential mechanisms for protecting chondrocytes. The relevant mechanisms of SK were analyzed by network pharmacology analysis. Chondrocytes were subjected to IL-1β intervention to simulate an inflammatory environment and received SK treatment. Then, anabolism, catabolism, and inflammatory markers were detected by western blot, qPCR, elisa, and immunofluorescence. Chondrogenic ability was evaluated by micromass and 3D culture assays. The rats were treated with destabilization of the medial meniscus (DMM) surgery to establish an OA model and SK intra-articular injections subsequently. Histological staining, immunohistochemistry, and micro-CT were performed to analyze the structural and morphological changes of cartilage and subchondral bone. In chondrocytes, IL-1β treatment reduced chondrogenic ability, promoted catabolism, and exacerbated inflammation by triggering the PI3K/AKT/NF-κB pathway, whereas SK treatment partially rescued these negative effects. In vivo, SK treatment effectively alleviated the degeneration of cartilage and subchondral bone, thereby delaying the progression of OA. In summary, SK alleviates chondrocyte inflammation and promotes chondrogenesis by inhibiting the PI3K/AKT/NF-κB pathway, thereby improving OA progression.

Osteoarthritis (OA), a joint disease that is associated with inflammatory processes is involved in joint destruction. Scutellarein (Scu), a component of the medicinal herbs Scutellaria barbata D. Don and Erigeron breviscapus (vant) Hand Mass, has anti-inflammatory effects. We explored the role of Scu in the development of OA and the underlying mechanisms. CCK-8 assays, Calcein-AM/PI and EdU staining were used to determine chondrocyte viability after Scu exposure. Western blot, qPCR, as well as ELISA were utilized to measure extracellular matrix (ECM) degradation and inflammation. Immunofluorescence (IF), western blot and luciferase assays were used to examine the NF-kappaB (NF-κB) pathway. Scu interacting proteins were predicted using network pharmacology analysis and molecular docking. X-ray, H&E, Safranin O-Fast Green(S-O), toluidine blue, and immunohistochemistry analysis were used to examine the therapeutic effects of Scu in OA using destabilization of medial meniscus (DMM) models. Scu demonstrated inhibitory effects on ECM degradation and pro-inflammatory factor levels in chondrocytes treated with IL-1β. Mechanistically, Scu inhibited the IL-1β-induced activation of the PI3K/Akt/ NF-κB signaling pathway cascades. Furthermore, Scu has been shown to have significant binding capacities to PI3K. Additionally, Scu ameliorated the OA progression in DMM models. Our findings suggest that Scu may contribute to the amelioration of OA progression by targeting the PI3K/Akt/NF-κB signaling pathway, implying Scu possesses promising therapeutic potential for the treatment of OA.

BACKGROUND:Isoliensinine is an active compound derived from Nelumbo nucifera which has long been used for its anti-inflammatory properties. However, the mechanism of Isoliensinine in the treatment of osteoarthritis is poorly known. PURPOSE:The present study aims to investigate whether Isoliensinine could alleviate osteoarthritis by regulating MAPK/NF-κB signaling pathway-mediated pyroptosis. METHODS:Network pharmacology and KEGG enrichment analysis were used to identify the therapeutic targets of Isoliensinine for OA. Molecular docking was used to confirm the binding ability of Isoliensinine and related proteins. In vitro, chondrocytes were stimulated with IL-1β to construct an inflammatory model and treated with Isoliensinine. The viability of the cells was assessed using the CCK-8 kit. The apoptosis rate of cells was measured using Annexin V-FITC/PI assay. And assessed the levels of ROS, lipid-ROS, and mitochondrial membrane potential. Corresponding assay kits were utilized to measure the levels of MDA and SOD. Subsequently, the anabolic and catabolic markers in chondrocytes, alongside inflammatory targets were measured by RT-PCR and Western blot. The expression level of pyroptosis and MAPK/NF-κB signaling pathway-related targets was examined. Furthermore, we constructed a rat osteoarthritis model using ACLT surgery. We then assessed the progression of osteoarthritis by Micro-CT, H&E staining, S&F staining and immunohistochemistry. RESULTS:Enrichment analysis showed that Isoliensinine treatment of osteoarthritis may be through the MAPK/NF-κB pathway, and molecular docking showed that Isoliensinine and MAPK/NF-κB pathway proteins had a good binding ability. Data showed that Isoliensinine could reduce ECM degradation and inflammation, and inhibit IL-1β-induced apoptosis. It also mitigated ROS and LPO activation, regulated mitochondrial dysfunction, and reduced intracellular oxidative stress levels. Furthermore, Western blot showed that Isoliensinine also inhibited the activation of the MAPK/NF-κB pathway, thereby inhibiting the pyroptosis of chondrocytes. In vivo, Micro-CT, H&E staining and S&F staining results showed that Isoliensinine could effectively improve joint damage caused by osteoarthritis. And IHC analyses indicated NLRP3, MMP3 protein expression were significantly diminished and Collagen II expression was increased in the Isoliensinine treatment groups. CONCLUSION:In conclusion, our study suggested that Isoliensinine mitigates ECM degradation, oxidative stress, chondrocytes apoptosis, and pyroptosis through the inhibition of the MAPK and NF-κB pathways, thereby delaying the progression of osteoarthritis.

Background:Osteoarthritis (OA) is the most common joint disease, which mainly damages articular cartilage and involves the whole joint tissue. It has the characteristics of long course, repeated symptoms and high disability rate, and the incidence trend is gradually increasing. Tetramethylpyrazine (TMP) is the main alkaloid active substance in Ligusticum wallichii, a traditional Chinese medicine, which has the effect of promoting blood circulation and dredging collaterals, and has a good effect on the treatment of early OA, but its molecular mechanism has not been fully clarified so far. Based on network pharmacology, molecular docking simulation and animal experiments, this study explored the target and molecular mechanism of TMP in the treatment of OA. Methods:We used PubChem, SwissTargetPrediction, and PharmMapper databases to predict the molecular structure and potential targets of TMP. GeneCards and DisGeNET databases were used to predict the relevant targets of OA. Apply UniProt database to convert targets into unified gene names, and proofread and remove duplicate gene names. The intersection targets of TMP and OA obtained on venny2.1.0 website were submitted to the STRING database to construct a PPI network. CytoScape 3.8.2 software was used to analyze the PPI network and obtain the sub-network modules and 10 key targets. The intersection targets of TMP and OA were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment using DAVID 6.8 database. The intersecting targets of TMP and OA, the biological process of GO enrichment, and KEGG signaling pathway were imported into Cytoscape 3.8.2 software to construct the TMP-target-pathway network diagram. Use molecular docking technology to simulate the interaction between TMP molecules and key targets, and predict the binding mode and binding ability. Animal models of rabbit knee osteoarthritis were prepared, and magnetic resonance imager (MRI) and fluorescence quantitative PCR (RT-qPCR) were used to observe the effect of TMP in treating OA as well as the expression of key target genes. Results:585 potential targets of TMP, 3,857 potential targets of OA, and 49 intersecting targets of TMP and OA were obtained. The top 10 key target genes were obtained, in order of ranking: ALB, ESR1, IL10, CAT, F2, MPO, C3, CYP3A4, CYP2C9, ANXA1. GO and KEGG analysis implied that the key targets might act on OA by affecting endothelial cell permeability, peri-articular microcirculatory status, NETs production, activation of complement system and coagulation pathway, regulation of immune function of macrophages and T cells, and substance metabolism pathway , etc. The molecular mechanism might involve the formation of neutrophil extracellular trap, regulation of the actin cytoskeleton, complement and coagulation cascades, and T cell receptor signaling pathways, etc. Molecular docking simulations showed that the binding energy of IL10 and ANXA1 to TMP was greater than -5Kal/mol, but the other key target proteins showed better binding to TMP, and the binding energy was less than -5 kcal/mol. Animal experiments showed that TMP had a significant therapeutic effect on OA. The TMP group had significantly reduced knee joint effusion and bone marrow damage compared to the OA group ( < 0.05). The qRT-PCR results showed that compared with the OA group, the mRNA expression of ESR1, CAT, C3, CYP3A4, CYP2C9, and ANXA1 in the TMP group increased ( < 0.05), while there was no significant difference in mRNA expression of ALB, IL-10, F2, MPO, etc. ( > 0.05). Conclusion:TMP is effective in the treatment of OA, with multi-target and multi-pathway interactions. ESR1, CAT, C3, CYP3A4, CYP2C9, and ANXA1 may be potential targets for TMP treatment of OA. The molecular mechanism mainly involves the formation of neutrophil extracellular trap, regulation of the actin cytoskeleton, complement and coagulation cascades, and T cell receptor signaling pathways, etc.

Inflammation and extracellular matrix (ECM) degradation are two major factors involved in the pathogenesis of osteoarthritis (OA). Wedelolactone, a natural compound classified as a coumestan, is isolated from the medicinal plants Eclipta alba and Wedelia calendulacea. In this study, we assessed the protective effects of Wedelolactone on chondrocytes in OA. Our findings show that pretreatment with Wedelolactone effectively inhibited the IL-1β-induced upregulation of COX‑2, iNOS, TNF-α, and IL6 in chondrocytes, contributing to inflammation suppression. Moreover, pretreatment with Wedelolactone followed by IL-1β treatment significantly increased the expression of Collagen II and SOX9, while decreasing the expression of Adamts5, MMP1, MMP3, and MMP13, thereby promoting ECM protection. Through Network pharmacology Analysis, we identified 14 key targets that link Wedelolactone and OA. GO and KEGG pathway analysis suggested that Wedelolactone primarily impacted OA by targeting inflammatory responses, particularly the NF-κB signaling pathway. Further studies demonstrated Wedelolactone prevented IL-1β-induced activation of NF-κB signaling pathway by inhibiting the translocation of p65 and the preventing the degradation of IκBα in human chondrocytes. Molecular docking studies also indicated that Wedelolactone can directly bind to the NF-κB complex, thereby inhibited the nuclear localization of p65. In vivo experiments demonstrated that Wedelolactone can alleviate cartilage damage in DMM mice model. In summary, Wedelolactone appears to mitigate inflammation and cartilage degeneration by suppressing the NF-κB signaling pathway, thereby alleviating OA progression. Our results suggested Wedelolactone may offer therapeutic advantages for OA treatment.

Osteoarthritis (OA) is the predominant cause of disability among elderly people worldwide and is characterized by cartilage degeneration and excessive bone formation. Phillyrin, derived from forsythia, is a key extract renowned for its pronounced antibacterial and anti-inflammatory effects. Forsythia, deeply integrated into traditional Oriental medicine, has historically been utilized for its various pharmacological effects, including antibacterial, anti-inflammatory, and hepato-protective properties. Nevertheless, the anti-inflammatory impact of phillyrin on the progression of osteoarthritis remains enigmatic. The objective of this research was to assess the anti-inflammatory and anti-aging properties of phillyrin in mouse chondrocytes induced by IL-1β, as well as to elucidate the fundamental mechanisms underlying the phenomenon at play. Additionally, the investigation extends to observing the impact of phillyrin by establishing a murine osteoarthritic model. The ultimate goal was to identify phillyrin as a potential antiosteoarthritic agent. This investigation employs a multifaceted approach. Initially, key action targets of phillyrin, along with its probable action pathways, were identified by molecular docking and network pharmacological techniques. These findings were subsequently confirmed through both in vivo and in vitro studies. Network pharmacological analysis revealed NFE2L2 (NRF2), NFKB1, TLR4, and SERPING1 as pivotal candidate targets for the treatment of osteoarthritis with phillyrin. Molecular docking revealed hydrogen bond interactions between phillyrin and Arg415, Arg483, Ser508, and Asn387 on the Nrf2 receptor, while electrostatic interactions occurred with residues Arg415 and Arg380. Experiments conducted in vitro indicated that phillyrin preconditioning hindered the IL-1β-induced expression of proinflammatory factors which included TNF-α, COX-2, IL-6, and iNOS. Furthermore, phillyrin counteracts the IL-1β-induced degradation of aggrecan and collagen II within the extracellular matrix (ECM). This protective action is caused by the inhibition of the NF-κB pathway by phillyrin. Additionally, the mitigation of chondrocyte aging by phillyrin was observed. Our investigation revealed that phillyrin mitigates inflammation and counteracts cartilage degeneration in osteoarthritis (OA) patients by suppressing inflammation in chondrocytes and impeding aging through suppression of the NF-κB pathway.

As a traditional medicinal food, Kudzu root (KR) has been proven to be an effective medicine for treating osteoporosis (OP). However, its precise targets and underlying integrated pharmacological mechanisms on OP have not yet been systematically investigated. The aim of the present study was to systemically explore the active ingredients, molecular targets, and ingredient-target network of KR against OP by the methods of network pharmacology followed by biological validation in a glucocorticoid-induced bone loss model of zebrafish. Our results identified a total of 15 active compounds with good pharmacokinetic properties in KR and 119 targets related to OP from correspondent databases, forming an ingredient-target network. Additionally, the protein-protein interaction (PPI) network further identified 39 core targets. Enrichment analyses with functional annotation revealed that the TNF signaling pathway and osteoclast differentiation process were significantly enriched by multi-targets including AKT1, P65, MAPK14, JUN, TNF-α, MMP9, IL6, and IL1B, etc., and served as the critical targets for molecular docking, molecular dynamics simulation, and in vivo experiment validation. These critical targets performed effectively in molecular docking and molecular dynamics, with AKT1, MMP9, and TNF-α exhibiting more prominent binding energy with Coumestrol, Genistein, and Genistein 7-glucoside, respectively. Further experimental validation in a zebrafish model indicated that KR could regulate the expressions of critical targets (AKT1, P65, MAPK14, JUN, TNF-α, and MMP9). This study provides a systemic perspective of the relationships between the active ingredients of KR and their multi-targets in OP, thereby constructing a pharmacological network to clarify the mechanisms by which KR ameliorates OP.

Background:Diabetic liver injury (DLI) is a common complication of diabetes mellitus (DM), which seriously endangers the health of diabetic patients. Puerarin, the main active component of , has shown positive effects in lowering blood glucose and lipids, resisting oxidative stress, and protecting the liver. However, the mechanism of protective effect of Puerarin on DLI remains unclear. Methods:Various databases were used to screen for targets of Puerarin, ferroptosis and DLI. Protein-protein interaction (PPI) network and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were used to predict key targets and pathways. Molecular docking was used to predict the interactions between Puerarin and core targets. KK/Upj-Ay/J (KKAy) mice and high glucose (HG)-induced AML12 cells were used to study the protective effect of Puerarin on DLI. The molecular mechanisms by which Puerarin acts were further verified by in vivo and in vitro experiments. Results:KEGG analysis indicated that the JAK/STAT pathway might be related to the anti-DLI effect of Puerarin. Molecular docking revealed that Puerarin has good affinity for JAK2 and STAT3. In vivo, Puerarin (80 mg/kg) reduced body weight, blood glucose, blood lipids and liver function in KKAy mice fed a high-sugar, high-fat diet. Puerarin also ameliorated hepatic pathological changes and inflammatory responses, and attenuated oxidative stress and iron overload in KKAy mice. Western blotting results showed that Puerarin could regulate the expression of proteins related to JAK2/STAT3 pathway and ferroptosis pathway. In vitro, Puerarin (25, 50, 100 μM) increased cell viability and decreased steatosis and liver function indexes in AML12 cells induced by HG (30 mm) to varying degrees. More importantly, AG490 blocker experiments showed that the regulation of ferroptosis process by Puerarin was dependent on the JAK2/STAT3 pathway. Conclusion:In conclusion, this study revealed Puerarin may regulate the ferroptosis process by inhibiting the JAK2/STAT3 pathway for the treatment of DLI.

Integrating network pharmacological analysis and bioinformatic techniques, this study systematically investigated the molecular mechanisms of six medicinal food homologous plants (, , , , , and ) against colorectal cancer. Through screening the TCMSP database, 303 active compounds and 453 drug targets were identified. By integrating differential expression gene analysis with WGCNA on the GSE41258 dataset from the GEO database, 49 potential therapeutic targets were identified. GO and KEGG enrichment analyses demonstrated that these targets are primarily involved in drug response, fatty acid metabolism, and key cancer-related pathways. Cross-validation using three machine learning algorithms-LASSO regression, SVM-RFE, and Random Forest-pinpointed four critical target genes: CA1, CCND1, CXCL2, and EIF6. Further, CIBERSORT immune infiltration analysis revealed strong associations between these core genes and the tumor immune microenvironment in colorectal cancer patients, notably in modulating M0 macrophage infiltration and mast cell activity. Molecular docking analyses confirmed robust binding interactions between active compounds and core target proteins. This study systematically elucidated the molecular mechanisms of six medicinal food homologous plants against colorectal cancer, providing scientific evidence for their rational clinical application.

Background:Knee osteoarthritis (KOA) is a persistent degenerative condition characterized by the deterioration of cartilage. The Chinese herbal formula Radix Rehmanniae Praeparata- Angelica Sinensis-Radix Achyranthis Bidentatae (RAR) has often been used in effective prescriptions for KOA as the main functional drug, but its underlying mechanism remains unclear. Therefore, network pharmacology and verification experiments were employed to investigate the impact and mode of action of RAR in the treatment of KOA. Methods:The destabilization of the medial meniscus model (DMM) was utilized to assess the anti-KOA effect of RAR by using gait analysis, micro-computed tomography (Micro-CT), and histology. Primary chondrocytes were extracted from the rib cartilage of a newborn mouse. The protective effects of RAR on OA cells were evaluated using a CCK-8 assay. The antioxidative effect of RAR was determined by measuring reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) production. Furthermore, network pharmacology and molecular docking were utilized to propose possible RAR targets for KOA, which were further verified through experiments. Results:In vivo, RAR significantly ameliorated DMM-induced KOA characteristics, such as subchondral bone sclerosis, cartilage deterioration, gait abnormalities, and the degree of knee swelling. In vitro, RAR stimulated chondrocyte proliferation and the expression of Col2a1, Comp, and Acan. Moreover, RAR treatment significantly reduced ROS accumulation in an OA cell model induced by IL-1β and increased the activity of antioxidant enzymes (SOD and GSH). Network pharmacology analysis combined with molecular docking showed that Mapk1 might be a key therapeutic target. Subsequent research showed that RAR could downregulate Mapk1 mRNA levels in IL-1β-induced chondrocytes and DMM-induced rats. Conclusion:RAR inhibited extracellular matrix (ECM) degradation and oxidative stress response via the MAPK signaling pathway in KOA, and Mapk1 may be a core target.