Pheretima aspergillum decoction suppresses inflammation and relieves asthma in a mouse model of bronchial asthma by NF-κB inhibition.
Huang Chuan-Qi,Li Wei,Wu Bo,Chen Wei-Ming,Chen Li-Hong,Mo Guan-Wen,Zhang Qiu-Feng,Gong Ling,Li Jing,Zhang Hong-Chao,Zhu Hai-Mei,Zeng Qing-Zhong
Journal of ethnopharmacology
ETHNOPHARMACOLOGICAL RELEVANCE AND AIM OF THE STUDY:Guang-Pheretima, the live form of the earthworm Pheretima aspergillum, is a traditional Chinese medicine commonly used for the treatment of asthma, cough, stroke, epilepsy and other diseases due to its anti-inflammatory, anti-asthmatic, anti-seizure, thrombolytic and diuretic properties. Although Guang-Pheretima is effective in the relief of asthma, its pharmacological activity and the underlying molecular mechanisms are not fully understood. Hence, we investigated the effects of a Pheretima aspergillum decoction (PAD) against inflammation in a model of ovalbumin (OVA)-induced asthma in BALB/c mice, as well as the nuclear factor-κB (NF-κB) pathway involved in this process. MATERIALS AND METHODS:OVA was used to sensitize and challenge the airway of the mice, and PAD was administrated by gavage. We measured airway hyperresponsiveness (AHR) in the mice 24h following a final methacholine challenge with whole-body plethysmography. The bronchoalveolar lavage fluid (BALF), serum and pulmonary tissues were collected 48h after the last challenge. The levels of inflammatory factors and the related mRNAs were determined by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR), respectively. The number of differential inflammatory cells in the BALF was counted. Serum total and OVA-specific IgE levels were measured with ELISA. The activation of NF-κB signaling in the lung was detected by western blotting. In addition, the lung tissues were stained with hematoxylin and eosin or periodic acid Schiff stain for histopathological examination. RESULTS:PAD treatment significantly alleviated AHR in the asthmatic mice, decreased the mRNA and protein levels of IL-4, IL-5 and IL-13 and downregulated IgE. In addition, PAD treatment attenuated mucus secretion and infiltration of inflammatory cells in the lung while inhibiting the activation of NF-κB signaling. CONCLUSIONS:PAD effectively inhibited the activation of NF-κB signaling in the lungs of mice with OVA-induced asthma, and mitigated AHR and Th2 type inflammatory reactions. Therefore, PAD may serve as a drug candidate for asthma treatment.
Pheretima aspergillum extract attenuates high-KCl-induced mitochondrial injury and pro-fibrotic events in cardiomyoblast cells.
Huang Pei-Chen,Shibu Marthandam Asokan,Kuo Chia-Hua,Han Chien-Kuo,Chen Yueh-Sheng,Lo Feng-Yueh,Li Heng,Viswanadha Vijaya Padma,Lai Chao-Hung,Li Xudong,Huang Chih-Yang
Hyperkalemia is often associated with cardiac dysfunction. In this study an earthworm extract (dilong) was prepared from dried Pheretima aspergillum powder and its effect against high-KCl challenge was determined in H9c2 cardiomyoblast cells. H9c2 cells pre-treated with dilong (31.25, 62.5, 125, and 250 mg/mL) for 24 hours, where challenged with different doses of KCl treatment for 3 hours to determine the protective mechanisms of dilong against cardiac fibrosis. High-KCl administration induced mitochondrial injury and elevated the levels of pro-apoptotic proteins. The mediators of fibrosis such as ERK, uPA, SP1, and CTGF were also found to be upregulated in high-KCl condition. However, dilong treatment enhanced IGF1R/PI3k/Akt activation which is associated with cell survival. In addition, dilong also reversed high-KCl induced cardiac fibrosis related events in H9c2 cells and displayed a strong cardio-protective effect. Therefore, dilong is a potential agent to overcome cardiac events associated with high-KCl toxicity.
Anti-inflammatory activities of Guang-Pheretima extract in lipopolysaccharide-stimulated RAW 264.7 murine macrophages.
Huang Chuanqi,Li Wei,Zhang Qiufeng,Chen Lihong,Chen Weiming,Zhang Hongchao,Ni Yuxin
BMC complementary and alternative medicine
BACKGROUND:Guang-Pheretima, which is originated from Pheretima aspergillum, has been documented in academic Chinese herbal studies for nearly 2000 years for its prominent treating effects of various inflammatory diseases such as asthma, cough and fever. However, the anti-inflammatory activity and mechanism of Guang-Pheretima has been rarely reported. Hence, we investigated the inhibitory effect and the underlying mechanism of Guang-Pheretima aqueous extracts on inflammatory response in RAW 264.7 cells. METHOD:RAW 264.7 macrophages were pretreated with various concentrations of Guang-Pheretima decoction (GPD) or protein-free Guang-Pheretima decoction (PF-GPD) and subsequently stimulated with lipopolysaccharide (LPS) to trigger the inflammatory response. Productions of nitric oxide (NO) were determined by Griess reaction, and prostaglandin E (PGE), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 were measured by enzyme-linked immunosorbent assays (ELISA). The protein expressions and messenger ribonucleic acid (mRNA) amounts of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were analyzed by Western Blot and Real-Time polymerase chain reaction (PCR), respectively. Finally, the translocation of nuclear factor (NF)-κB was observed by Western Blot. RESULTS:GPD of the experimental concentrations showed no anti-inflammatory activity. In contrast, PF-GPD at concentrations of 40-320 μg/mL significantly inhibited NF-κB activation and reduced the production of inflammatory mediators, such as NO, PGE, TNF-α, as well as the related key synthases including iNOS and COX-2. Moreover, PF-GPD markedly suppressed the release of inflammatory cytokines, such as IL-1β and IL-6. CONCLUSION:These results demonstrate the excellent anti-inflammatory properties of PF-GPD, and suggest that Guang-Pheretima may be used to treat and prevent certain inflammatory diseases.
Induction of apoptosis-like cell death by coelomocyte extracts from Eisenia andrei earthworms.
Mácsik Levente László,Somogyi Ildikó,Opper Balázs,Bovári-Biri Judit,Pollák Edit,Molnár László,Németh Péter,Engelmann Péter
Earthworm's innate immunity is maintained by cellular and humoral components. Our objective was to characterize the cytotoxicity leading to target cell death caused by earthworm coelomocytes. Coelomocyte lysates induced strong cytotoxicity in tumor cell lines. Transmission electron microscopy revealed cell membrane and intracellular damage in cells treated with coelomocyte lysates. Using TUNEL-assay, within 5 min of incubation we detected DNA fragmentation. Moreover, we found phosphatidylserine translocation in target cell-membranes. Furthermore, we detected dose-dependent Ca(2+) influx and decrease of mitochondrial membrane potential in coelomocyte lysate-treated cells. Interestingly, caspase 3/8 activation was undetectable in exposed tumor cells. One such cytotoxic molecule, lysenin identified in earthworms binds to sphingomyelin and causes target cell lysis in vertebrates. Pretreatment with our anti-lysenin monoclonal antibody rescued the majority but not all target cells from coelomocyte induced death. These data suggest that, not only lysenin but also other factors participate in the caspase-independent apoptosis induced by coelomocytes.
Structural basis for cooperative oxygen binding and bracelet-assisted assembly of Lumbricus terrestris hemoglobin.
Chen Wei-Ting,Chen Yu-Chuen,Liou Horng-Huei,Chao Chih-Yu
The iron-containing hemoglobins (Hbs) are essential proteins to serve as oxygen transporters in the blood. Among various kinds of Hbs, the earthworm Hbs are the champions in carrying oxygen due to not only their large size but also the unusually high cooperativity of ligand binding. However, the cooperative oxygen binding mechanisms are still mostly unknown. Here we report the cryo-electron microscopy structure of Lumbricus terrestris Hb in its native, oxygenated state at 9.1 Å resolution, showing remarkable differences from the carbon monoxide-binding X-ray structure. Our structural analysis first indicates that the cooperative ligand binding of L. terrestris Hb requires tertiary and quaternary transitions in the heme pocket and a global subunit movement facilitated by intra-ring and inter-ring contacts. Moreover, the additional sinusoidal bracelet provides the confirmation for the long-standing debate about the additional electron densities absent in the X-ray crystal structure.
Tabletting Process of Pellets Containing Bioactive Fraction DLBS1033F Isolated from Lumbricus rubellus: Challenge and Strategy.
Christy Gabriela,Tjandrawinata Raymond Rubianto,Rachmawati Heni
Biological & pharmaceutical bulletin
DLBS1033 is a mixture of bioactive proteins fractinated from the earthworm Lumbricus rubellus which possesses antithrombotic and thrombolytic activities. DLBS1033 contains a potent fibrinolytic enzyme with excellent specificity for fibrin. A commercial oral enteric coated tablet for administration 3 times daily is available on the market from the DLBS manufacturer. This research aimed to develop a multiparticulate sustained-release DLBS1033 Forte (DLBS1033F) tablet (with more potent fibrinolytic activity than DLBS1033), which has targeted release in the small intestine and colon. The chemical and physical stability of the fibrinolytic enzyme was expected to be preserved in these bowel segments. The sustained-release system was designed to maintain the effective plasma concentration of the proteins and prolong the thrombolytic activity. This was done by incorporating the proteins in a multiparticulate solid that was subsequently compressed into the monolithic system of a sustained-release tablet. The challenge was to maintain pellet integrity after the compression process. A set of studies confirmed the preserved physical shape of the pellets. In addition, in vitro release studies of the multiparticulate tablet in reassembling media of the gastrointestinal tract indicated the successful formulation of DLBS1033F as an enteric as well as sustained-release solid dosage form.
Statistical Correlations between NMR Spectroscopy and Direct Infusion FT-ICR Mass Spectrometry Aid Annotation of Unknowns in Metabolomics.
Hao Jie,Liebeke Manuel,Sommer Ulf,Viant Mark R,Bundy Jacob G,Ebbels Timothy M D
NMR spectroscopy and mass spectrometry are the two major analytical platforms for metabolomics, and both generate substantial data with hundreds to thousands of observed peaks for a single sample. Many of these are unknown, and peak assignment is generally complex and time-consuming. Statistical correlations between data types have proven useful in expediting this process, for example, in prioritizing candidate assignments. However, this approach has not been formally assessed for the comparison of direct-infusion mass spectrometry (DIMS) and NMR data. Here, we present a systematic analysis of a sample set (tissue extracts), and the utility of a simple correlation threshold to aid metabolite identification. The correlations were surprisingly successful in linking structurally related signals, with 15 of 26 NMR-detectable metabolites having their highest correlation to a cognate MS ion. However, we found that the distribution of the correlations was highly dependent on the nature of the MS ion, such as the adduct type. This approach should help to alleviate this important bottleneck where both 1D NMR and DIMS data sets have been collected.
Anti-elastase, anti-tyrosinase and matrix metalloproteinase-1 inhibitory activity of earthworm extracts as potential new anti-aging agent.
Azmi Nurhazirah,Hashim Puziah,Hashim Dzulkifly M,Halimoon Normala,Majid Nik Muhamad Nik
Asian Pacific journal of tropical biomedicine
OBJECTIVE:To examine whether earthworms of Eisenia fetida, Lumbricus rubellus and Eudrilus eugeniae extracts have elastase, tyrosinase and matrix metalloproteinase-1 (MMP-1) inhibitory activity. METHODS:The earthworms extract was screened for elastase, tyrosinase and MMP-1 inhibitory activity and compared with the positive controls. It was also evaluated for whitening and anti-wrinkle capacity. RESULTS:The extract showed significantly (P<0.05) good elastase and tyrosinase inhibition and excellent MMP-1 inhibition compared to N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid. CONCLUSIONS:Earthworms extract showed effective inhibition of tyrosinase, elastase and MMP-1 activities. Therefore, this experiment further rationalizes the traditional use of this worm extracts which may be useful as an anti-wrinkle agent.
Dilong prevents the high-KCl cardioplegic solution administration-induced apoptosis in H9c2 cardiomyoblast cells mediated by MEK.
Han Chien-Kuo,Kuo Wei-Wen,Shen Chia-Yao,Chen Tung-Sheng,Pai Peiying,Tsai Chang-Hai,Lo Feng-Yueh,Ju Da-Tong,Huang Chih-Yang
The American journal of Chinese medicine
Infusion of high-KCl cardioplegic solution (High-KCS) is the most common method used to induce asystole before cardiac surgery. However, our previous study showed the High-KCS can cause the apoptosis of cardiomyocytes in patients who were administered High-KCS prior to undergoing coronary artery bypass graft (CABG) to treat coronary artery disease (CAD). Therefore, it is urgent today to find a complementary medicine to reduce this damage. Dilong (earthworm) has been used as a traditional medicine in China for several thousand years, and extract from the dilong has been empirically used in Asia for the treatment of vascular disorders. In this study, we applied dilong extract to reduce myocardial cell damage from High-KCS infusion and further investigated the mechanisms. H9c2 cardiomyoblast cells were cultured in serum-free medium for 4 h and then treated with dilong at 31.25, 62.5, 125, and 250 mg/mL for 24 h, which was then followed by High-KCS treatment for 3 h to detect the protective mechanisms of dilong behind cardiomyocyte apoptosis and cardiac fibrosis. Cells were harvested for MTT assay, TUNEL assay, and western blot analysis. We found that High-KCS-induced cardiomyocyte apoptosis, enhanced the protein level of pro-apoptotic Bad, released cytochrome c, and activated caspase-3 in H9c2 cells. The IGF-I/IGF-IR/ERK pathway involved in non-cardiomyocyte proliferation, and the expression/activation of uPA, Sp-1 and CTGF, which are implicated in the development of cardiac fibrosis were up-regulated, but the Akt for cardiomyocyte survival was greatly deactivated in postcardioplegic H9c2 cardiomyoblast cells. However, dilong was highly protective and totally reversed the apoptosis and cardiac fibrosis effects induced by High-KCS. Chemical inhibitors P38 (SB203580), JNK (SP600125), MEK (U0126), IGF-1 (AG1024), and PI3K (LY294002) were applied to investigate which is the mediator for dilong attenuated High-KCS stimulated caspase 3 activation. MEK (U0126) inhibitor completely blocked dilong inhibited caspase 3 activation in High-KCS treated H9c2 cells. The MEK siRNA was further applied to knockdown MEK to confirm our finding. We found dilong worked through MEK to inhibit caspase 3 activity induced by High-KCS in H9c2 cells. Furthermore, we used the pure component of dilong, Lumbrokinase, to block the High-KCS effect. Using the microscope to observe the cell viability, we found Lumbrokinase could reverse the High-KCS effect. Lumbrokinase could also reduce the protein levels of caspase 8, caspase 9, and caspase 3, and enhance the survival related proteins PI3K/Akt and Bcl2. These results demonstrate that dilong could be used as a potential agent to block the side effects caused by High-KCS in CABG surgery patients.
[Experimental study on the expression of α-SMA and HMGB1 in silicotic fibrosismodel rats interfered by lumbricus].
Yu T,Li G H,Jia Y M,Lou Y,Gan X Y
Zhonghua lao dong wei sheng zhi ye bing za zhi = Zhonghua laodong weisheng zhiyebing zazhi = Chinese journal of industrial hygiene and occupational diseases
To observe the expression of alpha smooth muscle actin (α-SMA) and high mo-bility group protein B1 (HMGB1) in silicosis model rats interfered by lumbricus. 45 rats were ran-domly divided into the control group, model group and group interfered by lumbricus. The silicosis model rats were established. The group interfered by lumbricus were intragastric administered with lumbricus decoction by the 4 ml/kg dose. The control group and model group were ig administered with the equal amount of normal saline. Each group were killed 5 rats on the 7(th), 14(th) and 28(th) day. The lung tissues were stained with HE and Sirius red methods. The mRNA expressions of α-SMA and HMGB1 were determined with RT-PCR; The pro-tein levels of α-SMA and HMGB1 were determined with Western blotting. Compared with the control group, the expression levels of α-SMA and HMGB1mRNA and protein in lung tissue of model group were grad-ually increased in the 7(th), 14(th) and 28(th) days, the difference was statistically significant (< 0.01) . Compared with model group, the levels of α-SMA and HMGB1mRNA and protein in lung tissue of group interfered by lumbricus were gradually lowered in the 7th, 14th and 28th days, the difference was statistically significant (<0.05, <0.01) . Lumbricus inhibits the collagen deposition and the formation of silicosis pulmo-nary fibrosis, which may be related to the inhibition of HMGB1 expression and activation of α-SMA in lung tis-sue.
Effective combined water and sideband suppression for low-speed tissue and in vivo MAS NMR.
Mobarhan Yalda Liaghati,Struppe Jochem,Fortier-McGill Blythe,Simpson André J
Analytical and bioanalytical chemistry
High-resolution magic angle spinning (HR-MAS) NMR is a powerful technique that can provide metabolic profiles and structural constraints on intact biological and environmental samples such as cells, tissues and living organisms. However, centripetal force from fast spinning can lead to a loss of sample integrity. In analyses focusing on structural organization, metabolite compartmentalization or in vivo studies, it is critical to keep the sample intact. As such, there is growing interest in slow spinning studies that preserve sample longevity. In this study, for example, reducing the spinning rate from 2500 to 500 Hz during the analysis of a living freshwater shrimp increased the 100% survivability threshold from ~14 to 40 h. Unfortunately, reducing spinning rate decreases the intensity of the isotropic signals and increases both the intensity and number of spinning sidebands, which mask spectral information. Interestingly, water suppression approaches such as excitation sculpting and W5 WATERGATE, which are effective at higher spinning rates, fail at lower spinning rates (<2500 Hz) while simpler approaches such as presaturation are not able to effectively suppress water when the ratio of water to biomass is very high, as is the case in vivo. As such there is a considerable gap in NMR approaches which can be used to suppress water signals and sidebands in biological samples at lower spinning rates. This research presents simple but practically important sequences that combine PURGE water suppression with both phase-adjusted spinning sidebands and an analogue of TOSS termed TOSS.243. The result is simple and effective water and sideband suppression even in extremely dilute samples in pure water down to ~100 Hz spinning rate. The approach is introduced, described and applied to a range of samples including, ex vivo worm tissue, Daphnia magna (water fleas), and in vivo Hyalella azteca (shrimp).
Glossoscolex paulistus hemoglobin with fluorescein isothiocyanate: Steady-state and time-resolved fluorescence.
Barros Ana E B,Barioni Marina B,Carvalho Francisco A O,Ito Amando Siuiti,Tabak Marcel
International journal of biological macromolecules
Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been followed, in the presence of urea, using fluorescein isothiocyanate (FITC). Binding of FITC to HbGp results in a significant quenching of probe fluorescence. Tryptophan emission decays present four characteristic lifetimes: two in the sub-nanosecond/picosecond, and two in the nanosecond time ranges. Tryptophan decays for pure HbGp and HbGp-FITC systems are similar. In the absence of denaturant, and up to 2.5mol/L of urea, the shorter lifetimes predominate. At 3.5 and 6.0mol/L of urea, the longer lifetimes increase significantly their contribution. Urea-induced unfolding process is characterized by protein oligomeric dissociation and denaturation of dissociated subunits. FITC emission decays for FITC-HbGp system are also multi-exponential with three lifetimes: two in the sub-nanosecond and one in the nanosecond range with a value similar to free probe in buffer. Increase of urea concentration leads to increase of the longer lifetime contribution, implying the removal of the quenching observed for the native HbGp-FITC system. Anisotropy decays are characterized by two rotational correlation times associated to re-orientational motions of the probe relative to protein. Our results suggest that FITC bound to HbGp is useful to monitor denaturant effects on the protein.
Thermoresponsive Collagen/Cell Penetrating Hybrid Peptide as Nanocarrier in Targeting-Free Cell Selection and Uptake.
Oh Myungeun,Hu Chloe,Urfano Selina F,Arostegui Merlyn,Slowinska Katarzyna
The effective delivery of therapeutics and imaging agents to a selected group of cells has been at the forefront of biomedical research. Unfortunately, the identification of the unique cell surface targets for cell selection remains a major challenge, particularly if cells within the selected group are not identical. Here we demonstrate a novel approach to cell section relying on a thermoresponsive peptide-based nanocarrier. The hybrid peptide containing cell-penetrating peptide (CPP) and collagen (COLL) domains is designed to undergo coil-to-helix transition (folding) below physiological temperature. Because only the helical form undergoes effective internalization by the cells, this approach allows effective temperature-discriminate cellular uptake. The cells selected for uptake are locally cooled, thus enabling the carrier to fold and subsequently internalize. Our approach demonstrates a generic method as selected cells could differ from the adjacent cells or could belong to the same cell population. The method is fast (<15 min) and selective; over 99.6% of cells in vitro internalized the peptide carrier at low temperatures (15 °C), while less than 0.2% internalized at 37 °C. In vivo results confirm the high selectivity of the method. The potential clinical applications in mixed cell differentiation carcinoma, most frequently encountered in breast and ovarian cancer, are envisioned.
[Study on proteins in Guangdilong by nano LC/orbitrap fusion lumos HR-MS].
Dong Hong-Shuang,Zhang Jing-Xian,Hu Qing,Wang Yan-Chun,Sun Jian,Zhang Su,Yu Hong,Feng Rui,Mao Xiu-Hong,Ji Shen
Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica
In the present study,fresh Guangdilong( GD),originating from Pheretima aspergillum,was taken as the object. The total proteins from GD were firstly separated by SDS-PAGE according to their molecular weights and in-gel digestion was then performed.After that,the peptides were analyzed by nano LC/orbitrap fusion lumos high resolution mass spectrometry( nano LC/orbitrap fusion lumos HR-MS). Protein identification was implemented by comparison with Annelida. fasta database using Proteome Discoverer software.As a result,386 proteins were tentatively identified,including chain F,globin B chain,glyceraldehyde-3-phosphate dehydrogenase,fibrinolytic protein,and so on. Most of the proteins took part in cell structure and energy metabolism,and fibrinolytic protein and lombricine kinase might be related to fibrinolytic activity. Protein classification based on gene ontology was carried out using PANTHER and KEGG for metabolic pathway enrichment. The results indicated that these proteins were related to diverse signal transduction pathways,including metabolic pathways,central carbon metabolism,biosynthesis of amino acids,ribosome,glycolysis,citrate cycle( TCA cycle),and so on. This study would lay the foundation for the further research on the proteins in GD and also their functions.
[Effect of Dilong on expression of fibrogenic factors TGF-β1 and α-SMA in lung tissue of mice with pulmonary fibrosis].
Wang Hui-Hui,Meng Yan-Li,Yang Zhi-Min,Wang Xiao-Xi,Xu Hui-Xing,Wang Wei-Ming
Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica
The aim of this paper was to investigate the effect of Dilong( geosaurus) on the expressions of fibrotic factors TGF-β1 and α-SMA in bleomycin-induced pulmonary fibrosis mice. The binding ability of Dilong to fibrotic factor TGF-β1 was initially detected by Biacore technology and verified by in vivo pharmacodynamics. A total of 60 SPF C57 mice were randomly divided into 6 groups. Except the blank group( injecting 0. 08 m L·kg-1 sodium chloride in the trachea),the other five groups were given bleomycin( 4 mg·kg-1) to replicate the pulmonary fibrosis model. After 14 days of drug treatment,the expressions of TGF-β1 and α-SMA were detected by Masson staining,immunohistochemistry and RT-PCR. The results of Biacore experiment showed that the extract of Dilong was well bound to TGF-β1 protein in vitro,and the binding value reached 619. 3. Compared with the model group,Masson's results showed that cellulose deposition in high-dose,medium-dose and low-dose Dilong groups decreased to varying degrees. RT-PCR results showed that different doses of Dilong could reduce protein and mRNA expressions of TGF-β1 and α-SMA to a certain extent in a dose-dependent manner. In conclusion,Dilong could delay the process of pulmonary fibrosis by binding to target protein TGF-β1 and inhibiting the expression of α-SMA.
Bioactive protein fraction DLBS1033 containing lumbrokinase isolated from Lumbricus rubellus: ex vivo, in vivo, and pharmaceutic studies.
Tjandrawinata Raymond R,Trisina Jessica,Rahayu Puji,Prasetya Lorentius Agung,Hanafiah Aang,Rachmawati Heni
Drug design, development and therapy
DLBS1033 is a bioactive protein fraction isolated from Lumbricus rubellus that tends to be unstable when exposed to the gastrointestinal environment. Accordingly, appropriate pharmaceutical development is needed to maximize absorption of the protein fraction in the gastrointestinal tract. In vitro, ex vivo, and in vivo stability assays were performed to study the stability of the bioactive protein fraction in gastric conditions. The bioactive protein fraction DLBS1033 was found to be unstable at low pH and in gastric fluid. The "enteric coating" formulation showed no leakage in gastric fluid-like medium and possessed a good release profile in simulated intestinal medium. DLBS1033 was absorbed through the small intestine in an intact protein form, confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis. This result confirmed that an enteric coating formula using methacrylic acid copolymer could protect DLBS1033 from the acidic condition of the stomach by preventing the release of DLBS1033 in the stomach, while promoting its release when reaching the intestine. From the blood concentration-versus-time curve, (99m)Tc-DLBS1033 showed a circulation half-life of 70 minutes. This relatively long biological half-life supports its function as a thrombolytic protein. Thus, an enteric delivery system is considered the best approach for DLBS1033 as an oral thrombolytic agent.
Toxicity studies of a bioactive protein with antithrombotic-thrombolytic activity, DLBS1033.
Sukandar Elin Yulinah,Anggadireja Kusnandar,Sigit Joseph Iskendiarso,Adnyana I Ketut,Tjandrawinata Raymond R
Drug and chemical toxicology
DLBS1033 is a bioactive protein extract containing Lumbricus rubellus and has been known to have antithrombotic/thrombolytic activity. The present study was aimed to assess the safety aspect of DLBS1033 in a preclinical setting, which included observation on toxic signs after acute and repeated administrations, and the drug's effect on prenatal development and drug interaction. In acute toxicity study, a high dose level (16.2 g/kg) of DLBS1033 was well tolerated. In subchronic toxicity study, after the doses of 270, 540 and 1080 mg/kg of DLBS1033 per day, no mortality was observed and other parameters were all observed to be normal. In prenatal developmental toxicity, no observed adverse effect level (NOAEL) of DLBS1033 was observed at a moderate dose (540 mg/kg). Coadministration of DLBS1033 with clopidogrel or aspirin did not cause gastric lesions, except when all three drugs were coadministrated. Taken together, results of the present study suggested that DLBS1033 is safe for long-term administration, with a caution at a high dose used during pregnancy, and can be used in combination with one of the antiplatelet drugs.
Data on proteins of lysenin family in coelomocytes of and obtained by tandem mass spectrometry coupled with liquid chromatography.
Swiderska Bianka,Kedracka-Krok Sylwia,Plytycz Barbara
Data in brief
The data described are related to the article "Lysenin family proteins in earthworm coelomocytes - comparative approach" (B. Swiderska, S. Kedracka-Krok, T. Panz, A.J. Morgan, A. Falniowski, P.Grzmil, B. Plytycz, 2016) . Lysenin family proteins were identified based on unique peptides sequenced by tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) in lumbricid earthworms and , the latter with or without the MUG-like fluorophore. Lysenin and lysenin-related protein 2 (LRP-2, fetidin) were identified in all 9 investigated specimens of sp. LRP-1 was identified in 5 of 6 specimens of , while LRP-3 was present in 2 of 3 investigated specimens of . Here, the detailed characteristics of identified peptides unique to the particular members of lysenin family present in each particular earthworm specimen was provided. The information concerning mass to charge ratio, retention time, modifications and score of unique peptides was given.
Lumbrokinase attenuates myocardial ischemia-reperfusion injury by inhibiting TLR4 signaling.
Wang Yi-Hsin,Chen Ke-Min,Chiu Ping-Sung,Lai Shih-Chan,Su Hsing-Hui,Jan Ming-Shiou,Lin Chia-Wei,Lu Dah-Yuu,Fu Yuan-Tsung,Liao Jiuan-Miaw,Chang Jinghua Tsai,Huang Shiang-Suo
Journal of molecular and cellular cardiology
Lumbrokinase, a novel antithrombotic agent, purified from the earthworm Lumbricus rubellus, has been clinically used to treat stroke and cardiovascular diseases. However, inflammatory responses associated with the cardioprotective effect of lumbrokinase remain unknown. In this study, the signaling pathways involved in lumbrokinase-inhibited expressions of inflammation mediators were investigated in rats subjected to myocardial ischemia-reperfusion (I-R) injury. The left main coronary artery of anesthetized rats was subjected to 1h occlusion and 3h reperfusion. The animals were treated with/without lumbrokinase and the severities of I-R-induced arrhythmias and infarction were compared. Lumbrokinase inhibited I-R-induced arrhythmias and reduced mortality, as well as decreased the lactate dehydrogenase levels in carotid blood. Lumbrokinase also inhibited the enhancement of I-R induced expressions of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), and matrix metalloproteinase (MMP)-9 through toll-like receptor 4 (TLR4) signaling pathway. Moreover, our results demonstrated that stimulation with lumbrokinase decreases the phosphorylation of JNK, IκB, and NF-κB. These findings suggested that lumbrokinase is a potent cardioprotective drug in rats with I-R injury. The cardioprotective effects of lumbrokinase may be correlated with its inhibitory effect on the I-R-induced expressions of COX-2, iNOS and MMP-9, mediated by TLR4 signaling through JNK and NF-κB pathways.
Chromatographic Fingerprint and Quantitative Analysis of Commercial (Guang Dilong) and Its Adulterants by UPLC-DAD.
Sun Jie,Tian Fang,Zhang Ying,Wu Menghua,Mao Runqian,Le Zhiyong,Xu Dongjin,Cao Hui,Ma Zhiguo
International journal of analytical chemistry
Guang Dilong is a Traditional Chinese Medicine prepared from the dried body of (E. Perrier), a species of earthworm. However, preparations of Guang Dilong may be adulterated by other species and a method of quality control is needed. A method was developed to analyze and authenticate commercial Guang Dilong, utilizing ultra-high performance liquid chromatography (UHPLC) coupled with diode array detection (DAD). Equipment included an Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 m). The mobile phase consisted of acetonitrile and 0.01% formic acid, pumped at 0.3 mL/min. Wavelength detection was at 260 nm. Twenty-two batches of confirmed samples (reference) from different sources and 20 batches of adulterated samples were analyzed to establish a reference fingerprint for commercial Guang Dilong. Five peaks in the fingerprints of the reference batches were identified as characteristic; six characteristic peaks in the fingerprints of the adulterants were identified by comparing their retention time with those of the references. The total 42 batches of samples were compared with the reference fingerprint, and the fingerprints of the samples were similar. The UHPLC-DAD method can simultaneously determine the contents of six compounds (hypoxanthine, xanthine, uridine, inosine, guanosine, and adenosine) in the reference and adulterated batches. The six compounds showed good regression ( > 0.9999) within test ranges. The recovery (accuracy) was 98.25 to 101.68%, with relative standard deviation <2.67%. In summary, this UHPLC-DAD method combines chromatographic fingerprint with quantification analysis and could be readily used as an efficient quality control method for Guang Dilong.
Bioactive proteins and peptides isolated from Chinese medicines with pharmaceutical potential.
Wong Kam Lok,Wong Ricky Ngok Shun,Zhang Liang,Liu Wing Keung,Ng Tzi Bun,Shaw Pang Chui,Kwok Philip Chi Lip,Lai Yau Ming,Zhang Zhang Jin,Zhang Yanbo,Tong Yao,Cheung Ho-Pan,Lu Jia,Sze Stephen Cho Wing
Some protein pharmaceuticals from Chinese medicine have been developed to treat cardiovascular diseases, genetic diseases, and cancer. Bioactive proteins with various pharmacological properties have been successfully isolated from animals such as Hirudo medicinalis (medicinal leech), Eisenia fetida (earthworm), and Mesobuthus martensii (Chinese scorpion), and from herbal medicines derived from species such as Cordyceps militaris, Ganoderma, Momordica cochinchinensis, Viscum album, Poria cocos, Senna obtusifolia, Panax notoginseng, Smilax glabra, Ginkgo biloba, Dioscorea batatas, and Trichosanthes kirilowii. This article reviews the isolation methods, molecular characteristics, bioactivities, pharmacological properties, and potential uses of bioactive proteins originating from these Chinese medicines.
The identification of functional proteins from amputated lumbricus Eisenia fetida on the wound healing process.
Yang Yuwei,Hu Haicong,Wang Wenqi,Duan Xiaojie,Luo Shilin,Wang Xiongfei,Sun Yikun
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Earthworm has a long history of being used for medical purposes in many countries. This study investigated the therapeutic effects of earthworm extract (G-90') from head-regenerating tissue of Eisenia fetida on wound healing process in vitro and in vivo. Among three salting out parts (ES1-ES3) of G-90', ES2 displayed the significant wound healing ability via promoting proliferation of fibroblast and keratinocyte as well as stimulating the expression of fibroblast growth factor and vascular endothelial growth factor in wound area. The wound healing-specific proteins in ES2 were further analysed by "bottom-up" proteomic analytic method. Two proteins in ES2 were identified through "bottom-up" analysis, but their effects on wound healing process remains enigmatic. The bioactive proteins (ES2) in G-90' enhance the proliferative phase in acute wound healing process, providing a new concept for transforming this natural material for use in wound therapy.
Data on the potent fibrinolytic effects of the earthworm and the lugworm.
Dong Yuanyuan,Woo Young Min,Lee Young-Ho,Ahn Mee Young,Lee Dong-Guen,Lee Sang-Hyeon,Ha Jong-Myung,Park Chan-Il,Kim Andre
Data in brief
There is technology available for anti-thrombus with earthworms, but the procedure is complex and extracts protein with inferior purity. In order to develop a simplified process with a stronger purity of protease, we investigated the earthworm and lugworm. We purified water extracts cut off at 10 kDa of molecular weight using ultrafiltration because proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. We purified EW1 (raw earthworm extract), EW2 (molecular weight (m.w) > 10 kDa of earthworm extract), and EW3 (m.w < 10 kDa) from the earthworm. Likewise, we purified LW1 (wild lugworm extract), LW2 (m.w > 10 kDa), and LW3 (m.w < 10 kDa) from the lugworm. Using a fibrin assay, we found that fibrinolytic activity of the specimens had a rank order of clear zone diameter: EW2 > EW1 > EW3 > LW2 > LW1 > LW3. In particular, EW2 and LW2 showed a potent fibrinolytic effect in two different worm specimens. The protein content of each sample was detected as 2.34 (EW1), 3.03 (EW2), 2.80 (LW1), 3.71 (LW2) mg/ml respectively, and their molecular weights were measured using SDS-PAGE. The samples contained the following amounts of total fatty acids: EW1, 3.61%; EW2, 0.48%; LW1, 4.96%; and LW2, 0.23%. We developed a process to increase the thrombolytic effect with a higher purity protein. The study results demonstrate this procedure and provide basic data for developing an anti-thrombolytic agent.
Development of a method for the analysis of hormones and pharmaceuticals in earthworms by quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Bergé Alexandre,Vulliet Emmanuelle
Analytical and bioanalytical chemistry
The earthworm represents a kind of creature in contact with the soil surface and usually exposed to a variety of organic pollutants from human activities. Therefore, it can be considered as an organism of choice for identifying pollution or better understanding the input of contaminants in food chains in particular through the contributions of sludge. Moreover, the use of organisms such as soil invertebrates is to be developed for ecotoxicological risk assessment of pollutants. In this context, a simple, rapid and effective multi-residue method was developed for the determination of 31 compounds including 11 steroids, 14 veterinary antibiotics and 6 human contaminants (paracetamol, sulfamethoxazole, fluvoxamine, carbamazepine, ibuprofen, bisphenol A) in earthworm. The sample preparation procedure was based on a salting-out extraction with acetonitrile (QuEChERS approach) that was optimised with regard to the acetonitrile/water ratio used in the extraction step, the choice of the clean-up and the quantity of the matrix. The optimised extraction method exhibited recoveries that comprised between 44 and 98 % for all the tested compounds. The limits of detection of all compounds were below 14 ng g(-1) and the limits of quantification (LOQ) comprised between 1.6 and 40 ng g(-1) (wet weight). The method was therefore applied to determine the levels of pharmaceuticals and hormones in six earthworm samples collected in various soils. Concentrations up to 195 ng g(-1) for bisphenol A were determined, between a few nanograms per gram and 43.1 ng g(-1) (estriol) for hormones and between a few nanograms per gram and 73.5 ng g(-1) (florfenicol) for pharmaceuticals. Experiments were also conducted in laboratory conditions to evaluate the accumulation of the target substances by earthworm.
Proteomics study revealed altered proteome of Dichogaster curgensis upon exposure to fly ash.
Markad Vijaykumar L,Adav Sunil S,Ghole Vikram S,Sze Siu Kwan,Kodam Kisan M
Fly ash is toxic and its escalating use as a soil amendment and disposal by dumping into environment is receiving alarming attention due to its impact on environment. Proteomics technology is being used for environmental studies since proteins respond rapidly when an organism is exposed to a toxicant, and hence soil engineers such as earthworms are used as model organisms to assess the toxic effects of soil toxicants. This study adopted proteomics technology and profiled proteome of earthworm Dichogaster curgensis that was exposed to fly ash, with main aim to elucidate fly ash effects on cellular and metabolic pathways. The functional classification of identified proteins revealed carbohydrate metabolism (14.36%), genetic information processing (15.02%), folding, sorting and degradation (10.83%), replication and repair (3.95%); environmental information processing (2.19%), signal transduction (9.61%), transport and catabolism (17.27%), energy metabolism (6.69%), etc. in the proteome. Proteomics data and functional assays revealed that the exposure of earthworm to fly ash induced protein synthesis, up-regulation of gluconeogenesis, disturbed energy metabolism, oxidative and cellular stress, and mis-folding of proteins. The regulation of ubiquitination, proteasome and modified alkaline comet assay in earthworm coelomocytes suggested DNA-protein cross link affecting chromatin remodeling and protein folding.
A novel type of luciferin from the Siberian luminous earthworm Fridericia heliota: structure elucidation by spectral studies and total synthesis.
Petushkov Valentin N,Dubinnyi Maxim A,Tsarkova Aleksandra S,Rodionova Natalja S,Baranov Mikhail S,Kublitski Vadim S,Shimomura Osamu,Yampolsky Ilia V
Angewandte Chemie (International ed. in English)
The structure elucidation and synthesis of the luciferin from the recently discovered luminous earthworm Fridericia heliota is reported. This luciferin is a key component of a novel ATP-dependent bioluminescence system. UV, fluorescence, NMR, and HRMS spectroscopy studies were performed on 0.005 mg of the isolated substance and revealed four isomeric structures that conform to spectral data. These isomers were chemically synthesized and one of them was found to produce light when reacted with a protein extract from F. heliota. The novel luciferin was found to have an unusual extensively modified peptidic nature, thus implying an unprecedented mechanism of action.
Lumbrokinase from earthworm extract ameliorates second-hand smoke-induced cardiac fibrosis.
Lai Chao-Hung,Han Chien-Kuo,Shibu Marthandam Asokan,Pai Pei Ying,Ho Tsung-Jung,Day Cecilia Hsuan,Tsai Fuu-Jen,Tsai Chang-Hai,Yao Chun-Hsu,Huang Chih-Yang
Exposure to tobacco smoke has epidemiologically been linked to the occurrence of cardiovascular disease among nonsmokers but the associated molecular events are not well elucidated yet. When Sprague Dawley rats were exposed to second-hand tobacco cigarette smoke twice a day for a 30 days period at an exposure rate of 10 cigarettes/30 min, they showed adverse effects including reduced left ventricle weight, increased cardiac damages, deteriorated cardiac features, and cardiac fibrosis. Exposure to second-hand smoking (SHS) increased the molecular markers of cardiac fibrosis such as urokinase plasminogen activator and matrix metallopeptidases. The modulations in the protein levels were led by the activation of extracellular signal-regulated kinases (ERK1/2), the transcription factor-specificity protein 1 (SP1), and the fibrogenic master switch-connective for epithelial-mesenchymal transition tissue growth factor there by indicating their effective role in SHS-induced myocardial infraction. Dilong, an edible earthworm extract used in Chinese medicine and its bioactive fibrinolytic enzyme product-lumbrokinase, when administered in rats, restricted the SHS exposure induced cardiac fibrosis and provided cardio-protection. The results show that lumbrokinase and dilong administration can efficiently prevent epidemiological incidence of cardiac disease among SHS-exposed nonsmokers.
FT-IR spectroscopy as a sentinel technology in earthworm toxicology.
Aja M,Jaya M,Vijayakumaran Nair K,Joe I Hubert
Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy
FT-IR spectroscopy is a useful tool for determining the biomolecular profile of micro-samples of body fluids such as coelomic fluid of earthworms. The present study focuses on the usefulness of the earthworm (Perionyx sansibaricus) coelomic fluid for observing pathologically induced biochemical changes. Compared to controls, appreciable changes in expression of peaks were observed in worms exposed to seven selected xenobiotics (pesticides, heavy metals, herbicides and detergents). Observation of bands in the region 1600-1690 cm(-1) indicates the presence of amide I band in all the worms. The peak at 2364 cm(-1) present as a weak band on day 7 of treatment, is shifted to 2358/2359 cm(-1) and more pronounced in most of the treated groups on day 14. Presence of band at 1663 cm(-1) in controls is attributed to CO stretching vibration representing the amino acid, glutamic acid. Under toxicological conditions vibration in this region is absent. Presence of the amino acid arginine (1633 cm(-1)) and lysine (1629 cm(-1)) and absence of glutamic acid (1663 cm(-1)) under toxicological stress were characteristic. FT-IR spectra of the coelomic fluid were similar under the sublethal and lethal concentrations of the test chemicals. The potential use of FT-IR spectral information as baseline data for toxicological studies and for monitoring the quality of the environment is recommended.
Antiproliferative Effect of Earthworm Coelomic Fluid of , , and on Squamous Cell Carcinoma-9 Cell Line: A Pilot Study.
Augustine Dominic,Rao Roopa S,Anbu Jayaraman,Chidambara Murthy K N
Introduction:The earthworm coelomic fluid (ECF) has shown proven antiproliferative effect against breast, liver, gastrointestinal, and brain cancer, but it is least explored in oral cancer. The present study is an attempt to investigate the antiproliferative activity of ECF on oral cancer cell line squamous cell carcinoma (SCC)-9. Materials and Methods:ECF was collected from the species (EE), (EF), and (PE) stored at -80°C. Percentage inhibition of ECF on squamous cell carcinoma-9 cells was recorded at 24 h. Protein estimation was done using Bradford protein assay validated by the biuret method. Cytotoxicity was tested at 2.5, 5, 10, 20, 40, and 80 μg/ml concentrations by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay in SCC-9 cells . GraphPad Prism 7.0 software was used to calculate the inhibitory concentration (IC). Chi-square test was used to analyze the difference between samples. Results:The test samples EE, EF, and PE inhibited the growth of SCC-9 cells significantly in a dose-dependent manner, and the IC values were found to be 4.6, 44.69, and 5.27 μg/ml, respectively. The antiproliferative effect was found to be variable among the three earthworm species with EE showing the most promising effect followed by PE and EF. Conclusion:Establishing the antiproliferative effect of ECF on oral cancer cells could be an initial step toward drug development and future anticancer research. The preliminary investigation has shown that ECF has a promising antiproliferative effect on oral cancer cells . SUMMARY:The present pilot study evaluated the antiproliferative effect of earthworm coelomic fluid (ECF) of (EE), (EF), and (PE) on squamous cell carcinoma-9 cell line. The ECF inhibitory activity was promising at inhibitory concentration values of 4.6, 44.69, and 5.27 μg/ml, respectively. Further studies pertaining to antiproliferative mechanism of EE, EF, and PE have been planned. ECF: Earthworm coelomic fluid, EE: , EF: , PE: , MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, SCC: Squamous cell carcinoma, BSA: Bovine serum albumin, PBS: Phosphored buffered saline, ATCC: American Type Culture Collection.
Purification and function of two analgesic and anti-inflammatory peptides from coelomic fluid of the earthworm, Eisenia foetida.
Li Chunlong,Chen Mengrou,Li Xiaojie,Yang Meifeng,Wang Ying,Yang Xinwang
The potential application of anti-inflammatory and analgesic compounds in medication and therapeutic care have become of increasing interest. We purified and characterized two novel analgesic and anti-inflammatory peptides, VQ-5 and AQ-5, from the coelomic fluid of the earthworm (Eisenia foetida). Their primary structures were determined as VSSVQ and AMADQ, respectively. Both peptides, especially AQ-5, exhibited analgesic activity in mouse models of persistent neuropathic pain and inflammation. AQ-5 also inhibited tumor necrosis factor alpha and cyclooxygenase-2 production. The mitogen-activated protein kinase signaling pathway, which is involved in analgesic and anti-inflammatory functions, was inhibited by AQ-5. Thus, the analgesic and anti-inflammatory effects of these peptides, especially AQ-5, demonstrated their potential as candidates for the development of novel analgesic medicines.
A novel role for earthworm peptide Lumbricusin as a regulator of neuroinflammation.
Seo Minchul,Lee Joon Ha,Baek Minhee,Kim Mi-Ae,Ahn Mi-Young,Kim Seong Hyun,Yun Eun-Young,Hwang Jae-Sam
Biochemical and biophysical research communications
Recently, we reported that Lumbricusin, an antimicrobial peptide isolated from earthworm Lumbricus terrestris, enhanced neuronal proliferation and ameliorated motor dysfunction and dopaminergic neurodegeneration. Accumulating evidence suggests that neurodegeneration is the primary pathological feature of acute or chronic inflammation mediated by microglia, the resident macrophage of the central nervous system. Therefore, microglial activation inhibitors may be useful as therapeutic agents for neurodegenerative diseases. To determine whether Lumbricusin ameliorates neuroinflammation through inhibition of microglial activation by lipopolysaccharides (LPS), we newly synthesized 9-mer Lumbricusin analogues based on the amino acid sequence of Lumbricusin. One of these, Lumbricusin Analogue 5 (LumA5; QLICWRRFR-NH), markedly reduced expression of enzymes (COX-2, iNOS), cytokines (IL-6, IL-1β, TNF-α), and signal transduction factors (AKT, MAPKs, NF-κB) involved in inflammation triggered by LPS in vitro and in vivo. In addition, LumA5 inhibited the cytotoxicity of conditioned medium prepared by LPS-activated BV-2 microglia to neuronal SH-SY5Y cells and improved cell viability. These results indicate that LumA5 may be a potential therapeutic agent for the treatment of various neuroinflammatory conditions.
Impact of LPS-induced cardiomyoblast cell apoptosis inhibited by earthworm extracts.
Li Ping-Chun,Tien Yun-Chen,Day Cecilia Hsuan,Pai Peiying,Kuo Wei-Wen,Chen Tung-Sheng,Kuo Chia-Hua,Tsai Chang-Hai,Ju Da-Tong,Huang Chih-Yang
Dilong is an earthworm extract with a dense nutritional content, widely used in Chinese herbal medicine to remove stasis and stimulate wound healing. Earthworm extracts are traditionally used by indigenous people throughout the world. How this Dilong inhibits Lipopolysaccharide (LPS)-induced cardiomyoblast cell apoptosis is still unclear. This study investigates the Dilong extract effect on LPS-induced apoptosis in H9c2 cardiomyoblast cells. LPS (1 μg/ml) administration for 24 h induced apoptosis in H9c2 cells. Cell apoptosis was detected using MTT, LDH, TUNEL assay and JC-1 staining. Western blot analysis was used to detect pro-apoptotic and anti-apoptotic proteins. Dilong extract totally blocked the LPS impact, leading to the activation of anti-apoptotic proteins, Bcl-2 and Bcl-xL, stabilized the mitochondria membrane and down-regulated the extrinsic and intrinsic pro-apoptotic proteins, TNF-α, active caspase-8, t-Bid, Bax, active caspase-9 and active caspase-3. Dilong could potentially serve as a cardio protective agent against LPS-induced H9c2 cardiomyoblast cell apoptosis.
Anti-Candida albicans effect of the protein-carbohydrate fraction obtained from the coelomic fluid of earthworm Dendrobaena veneta.
Fiołka Marta J,Czaplewska Paulina,Macur Katarzyna,Buchwald Tomasz,Kutkowska Jolanta,Paduch Roman,Kaczyński Zbigniew,Wydrych Jerzy,Urbanik-Sypniewska Teresa
An antifungal active fraction (AAF) from the coelomic fluid (CF) of the earthworm Dendrobaena veneta was isolated. The aim of the study was to analyze the antifungal activity of the AAF and to carry out chemical characterization of the fraction. The active fraction showed antifungal activity against a clinical C. albicans isolate, C. albicans ATCC 10231, and C. krusei ATCC 6258. It effectively reduced the metabolic activity of C. albicans cells and influenced their morphology after 48 hours of incubation. Scanning electron microscopy (SEM) images revealed loss of integrity of the cell wall induced by the active fraction. Calcofluor White staining showed changes in the structure of the C. albicans cell wall induced by the AAF. The fungal cells died via apoptosis and necrosis after the treatment with the studied fraction. Electrophoresis under native conditions revealed the presence of two compounds in the AAF, while SDS/PAGE gel electrophoresis showed several protein and carbohydrate compounds. The active fraction was analyzed using Raman spectroscopy, MALDI TOF/TOF, and ESI LC-MS. The Raman analysis confirmed the presence of proteins and determined their secondary structure. The MALDI TOF/TOF analysis facilitated detection of four main compounds with a mass of 7694.9 m/z, 12292.3 m/z, 21628.3 m/z, and 42923.2 m/z in the analyzed fraction. The presence of carbohydrate compounds in the preparation was confirmed by nuclear magnetic resonance (NMR) and gas chromatography (GC-MS). The ATR-FTIR spectrum of the AAF exhibited high similarity to the spectrum of egg white lysozyme. The AAF showed no endotoxicity and cytotoxicity towards normal skin fibroblasts (HSF); therefore, it can be used for the treatment of skin and mucous membrane candidiasis in the future. Given its efficient and selective action, the fraction seems to be a promising preparation with antifungal activity against C. albicans.
Earthworm extract attenuates silica-induced pulmonary fibrosis through Nrf2-dependent mechanisms.
Yang Jingjin,Wang Ting,Li Yan,Yao Wenxi,Ji Xiaoming,Wu Qiuyun,Han Lei,Han Ruhui,Yan Weiwen,Yuan Jiali,Ni Chunhui
Laboratory investigation; a journal of technical methods and pathology
Silicosis is an occupational pulmonary fibrosis caused by inhalation of silica (SiO) and there are no ideal drugs to treat this disease. Earthworm extract (EE), a natural nutrient, has been reported to have anti-inflammatory, antioxidant, and anti-apoptosis effects. The purpose of the current study was to test the protective effects of EE against SiO-induced pulmonary fibrosis and to explore the underlying mechanisms using both in vivo and in vitro models. We found that treatment with EE significantly reduced lung inflammation and fibrosis and improved lung structure and function in SiO-instilled mice. Further mechanistic investigations revealed that EE administration markedly inhibited SiO-induced oxidative stress, mitochondrial apoptotic pathway, and epithelial-mesenchymal transition in HBE and A549 cells. Furthermore, we demonstrate that Nrf2 activation partly mediates the interventional effects of EE against SiO-induced pulmonary fibrosis. Our study has identified EE to be a potential anti-oxidative, anti-inflammatory, and anti-fibrotic drug for silicosis.
[Protective effect of earthworm active ingredients against endoplasmic reticulum stress-induced acute liver injury in mice].
Zhao Ya-Fei,Gao Yang,Wu Xin-Fang,Wang Jian-Gang,Duan Leng-Xin
Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica
To study the protective effect of earthworm active ingredients(EWAs) against endoplasmic reticulum stress(ERS)-induced acute liver injury in mice. The model of liver injury was induced through intraperitoneal injection of 10%CCl4. Serum glutamic-pyruvic transaminase(ALT), glutamic-oxaloacetic transaminase(AST), superoxide dismutase(SOD) and glutathione peroxidase(GSH-PX) activity and malondialdehyde(MDA) concentration were detected by colorimetric method. Histological examination was performed through hematoxylin-eosin staining and light microscopy, and apoptosis was detected using terminal transferase dUTP nick end labeling. The expressions of ERS related proteins, including glucose regulated protein 78(GRP78), protein kinase R-like ER kinase(PERK), eukaryotic transcription initiation factor 2α(eIF2α), active transcription factor-4(ATF4) and CCAAT/enhancer binding homologous protein(CHOP), were measured by immunohistochemistry and Western blot. According to the results, compared with the model group,serological indexes in the high, middle and low doses of EWAs were significantly improved (P<0.05 or P<0.01), the extent of liver lesion was decreased and the degree of injury was significantly reduced, and that the liver index and the spleen index of mice were significantly changed(P<0.05 or P<0.01). In liver tissue, the expressions of GRP78 and CHOP were significantly decreased(P<0.05 or P<0.01). The protein expressions of GRP78, CHOP and its upstream signaling pathway PERK-eIF2-ATF4 were significantly decreased in each dose group(P<0.05 or P<0.01). In summary, EWAs has a significant protective effect on ERS-induced acute liver injury, and its mechanism may be correlated with the inhibition of oxidative stress and ERS, and down-regulation of ERS marker protein CHOP expression, andinhibition of apoptosis.
Soluble calcium-binding proteins (SCBPs) of the earthworm Lumbricus terrestris: molecular characterization and localization by FISH in muscle and neuronal tissue.
Thiruketheeswaran Prasath,Kiehl Ernst,D'Haese Jochen
Histochemistry and cell biology
Soluble calcium-binding proteins (SCBPs) of invertebrates probably serve like their vertebrate counterpart-the parvalbumins-as soluble relaxing factors in muscles. Three SCBP isoforms (SCBP) have been isolated and biochemically characterized in the earthworm Lumbricus terrestris (Huch et al. in J Comp Physiol B 158:325-334, 1988). For SCBP, we found two isoforms named SCBP. Both of them together with SCBP are present in the body wall muscle. In the gizzard solely, SCBP and no SCBP or SCBP could be detected. The coding sequences of all three isoforms consist of 534 bp for 178 amino acids and contain four EF-hand motifs, of which the second EF-hands are truncated. Recombinant proteins show heat stability and a Ca-dependent mobility shift similar to the native proteins, indicating comparable calcium-binding properties. All three isoforms are encoded by three distinct and differentially expressed genes. The genes for SCBP, SCBP, and SCBP are interrupted by only one intron, inserting at nearly the same positions. Northern blot analysis revealed two mRNA transcripts for SCBP of approximately 1250 and 1500 kb and one transcript for SCBP of approximately 1250 kb. SCBP mRNA was localized by fluorescent in situ hybridization in the body wall and the gizzard. The distribution of the staining intensities resembles that for the myosin ATPase activity and indicates a correlation between the amount of SCBP and speed of muscle contraction. In addition, SCBP mRNA was localized within the nervous tissue, the cerebral and subesophageal ganglia and the ventral nerve cord.
Determination of selected polychlorinated biphenyls in soil and earthworm (Eisenia fetida) using a QuEChERS-based method and gas chromatography with tandem MS.
He Zeying,Wang Lu,Peng Yi,Luo Ming,Wang Wenwen,Liu Xiaowei
Journal of separation science
Soil and earthworms are important objects in soil pollution assessment and environmental behavior and toxicity study for polychlorinated biphenyls. Accelerated solvent extraction and solid-phase extraction are generally required for the extraction and clean-up of polychlorinated biphenyls in soil and earthworm, which are tedious and time-consuming. In this work, a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure combined with gas chromatography and triple quadrupole mass spectrometry was developed for the determination of 20 selected polychlorinated biphenyl congeners in soil and earthworm. Different extraction times, solvents, and clean-up adsorbents were compared and optimized. The average recoveries from spiked soils ranged between 70 and 120% with satisfactory relative standard deviations for all the polychlorinated biphenyls. In earthworm, the recoveries of polychlorinated biphenyls 180, 183, and 189 were relatively low (< 70% in some spiking levels) compared to that of the other polychlorinated biphenyls. The limits of quantification were in the range of 0.01-0.05 ng/g. The method was successfully applied to the analysis of 66 agricultural soils. To our knowledge, a combined method based on QuEChERS for the determination of polychlorinated biphenyls in soil and earthworms has not been published before. The procedure proved to be simple, sensitive, efficient, and environmentally friendly.
The protective effect of the earthworm active ingredients on hepatocellular injury induced by endoplasmic reticulum stress.
Wang Qi,Duan Leng-Xin,Xu Zheng-Shun,Wang Jian-Gang,Xi Shou-Min
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
The earthworm is a widely used Chinese herbal medicine. There are more than 40 prescriptions including earthworms in the "Compendium of Materia Medica". TCM theory holds that earthworms exert antispasmodic and antipyretic effects through the liver meridian to calm the liver. However, the clinical effect of earthworms on liver injury has not been clearly demonstrated. We have previously established a method to extract the active ingredients from earthworms (hereinafter referred to as EWAs) . In the present study, we observed protective effect of the EWAs on tunicamycin-induced ERS (endoplasmic reticulum stress) model in human hepatic L02 cells. The results showed that the EWAs promote proliferation and reduced apoptosis of ERS model in L02 cells (P<0.01). The up-regulation of ERS-related proteins, including PERK (protein kinase RNA-like endoplasmic reticulum kinase), eIF2a (eukaryotic translation initiation factor 2a), ATF4 (activating transcription factor 4) and CHOP (CCAAT/enhancer binding protein homologous protein), in L02 cell under ERS was inhibited by treatment of the EWAs (P<0.01). In summary, our data suggest the EWAs can significant attenuate ERS-induced hepatocyte injury via PERK-eIF2a-ATF4 pathway.
Soluble calcium-binding proteins (SCBPs) of the earthworm Lumbricus terrestris: possible role as relaxation factors in muscle.
Thiruketheeswaran Prasath,Huch Ralf,D'Haese Jochen
Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology
The soluble Ca-binding protein (SCBP) from the earthworm Lumbricus terrestris was analyzed with regard to its role as a soluble muscle relaxation factor. The actomyosin ATPase activity was inhibited by the addition of decalcified SCBP as it binds Ca stronger than the regulatory proteins associated with the actomyosin. Competitive Ca-binding assays with decalcified actomyosin and SCBP showed that Ca is first bound to actomyosin and is subsequently taken over by SCBP with increasing incubation time. Ca competition experiments carried out with Ca loaded SCBP and fragmented sarcoplasmic reticulum vesicles revealed that Ca bound to SCBP can be deprived by the ATP-dependent Ca uptake of the sarcoplasmic reticulum. Furthermore, experiments in a diffusion chamber showed that the addition of SCBP significantly enhances the Ca flux in a concentration dependent manner. The amount of the Ca flux increase tends to reach a maximum value of about 70%. With all protein components isolated from the obliquely striated muscle, our in vitro experiments consistently show that SCBP may accelerate muscle relaxation similar as assumed for vertebrate parvalbumin.
Antitumor activity and apoptotic action of coelomic fluid from the earthworm Dendrobaena veneta against A549 human lung cancer cells.
Fiołka Marta J,Rzymowska Jolanta,Bilska Sylwia,Lewtak Kinga,Dmoszyńska-Graniczka Magdalena,Grzywnowicz Krzysztof,Kaźmierski Wojciech,Urbanik-Sypniewska Teresa
APMIS : acta pathologica, microbiologica, et immunologica Scandinavica
It is known that earthworm coelomic fluid (CF) can affect not only cancer but also normal cells. The study demonstrated that the CF of the earthworm Dendrobaena veneta exhibited cytotoxicity against A549 lung cancer cells but did not toward the bronchial epithelial cell line BEAS-2B. The selective effect on the tumor cells was achieved after a short-term CF heat pre-treatment at 70 °C. The cytotoxic effect of the CF was time- and concentration-dependent. The CF noticeably decreased the viability and affected the morphology of the A549 cells. Scanning electron microscopy revealed a different degree of destruction of the nucleus and cytoplasm of A549 cells. As determined by atomic force microscopy, the cell surface roughness increased while the cell stiffness was reduced upon the CF treatment. A twofold increase in the caspase 3, 4, 5, and 10 levels was observed in the A549 cells after the incubation with the CF. The results obtained by flow cytometry using Annexin V confirmed the proapoptotic effect of the earthworm CF on A549 lung cancer cells. The D. veneta CF and active fraction obtained with cytotoxicity toward A549 lung cancer is an interesting and promising preparation for further biological, chemical, and biomedical research.
An intelligentized strategy for endogenous small molecules characterization and quality evaluation of earthworm from two geographic origins by ultra-high performance HILIC/QTOF MS(E) and Progenesis QI.
Zhang Jingxian,Yang Wenzhi,Li Shangrong,Yao Shuai,Qi Peng,Yang Zhou,Feng Zijin,Hou Jinjun,Cai Luying,Yang Min,Wu Wanying,Guo De-An
Analytical and bioanalytical chemistry
Animal-derived medicines have been a vital component for traditional Chinese medicine. However, their quality control remains challenging due to the large polarity of the contained endogenous small molecules (ESMs) that are difficult to separate by reversed-phase HPLC. Herein, an intelligentized strategy by ultra-high performance hydrophilic interaction chromatography/quadrupole time-of-flight mass spectrometry (HILIC/QTOF-MS(E)) is presented, and used for the ESMs characterization and differentiation of two geographic origins of earthworm (Guang Di-long, GD; Hu Di-long, HD) as a case study. Chromatographic separation was performed on a BEH Amide column (2.1 × 100 mm, 1.7 μm). The MS(E) data in both negative and positive ion modes were acquired to record the high-accuracy MS and MS/MS data of all precursor ions. Automatic data processing was enabled by use of Progenesis QI software. As a consequence, 926 metabolites among 4705 features and 761 among 3418 features were characterized in the negative and positive modes, respectively, by searching the human metabolome database (HMDB). To reduce the false positive identifications, structural confirmation was conducted by comparison with the reference standards (tR and MS, MS/MS data) or matching with theoretical data or commercial library. Principal component analysis (PCA) of the GD and HD samples showed distinct classifications. Further orthogonal partial least squares discriminant analysis (OPLS-DA) and variable importance in projection (VIP) plot revealed the potential discriminatory markers between GD and HD. The present study provides a powerful and practical strategy that facilitates the primary metabolites characterization and quality evaluation of animal-derived medicines more efficiently. Graphical Abstract A general flowchart illustrating the application of ultra-high performance HILIC/QTOFMS(E) coupled with data processing by Progenesis QI to characterization of the endogenous small molecules and discriminatory analysis of animal-derived traditional medicine: earthworm as a case study.
Earthworm (Pheretima aspergillum) extract stimulates osteoblast activity and inhibits osteoclast differentiation.
Fu Yuan-Tsung,Chen Kuo-Yu,Chen Yueh-Sheng,Yao Chun-Hsu
BMC complementary and alternative medicine
BACKGROUND:The potential benefits of earthworm (Pheretima aspergillum) for healing have received considerable attention recently. Osteoblast and osteoclast activities are very important in bone remodeling, which is crucial to repair bone injuries. This study investigated the effects of earthworm extract on bone cell activities. METHODS:Osteoblast-like MG-63 cells and RAW 264.7 macrophage cells were used for identifying the cellular effects of different concentrations of earthworm extract on osteoblasts and osteoclasts, respectively. The optimal concentration of earthworm extract was determined by mitochondrial colorimetric assay, alkaline phosphatase activity, matrix calcium deposition, Western blotting and tartrate-resistant acid phosphatase activity. RESULTS:Earthworm extract had a dose-dependent effect on bone cell activities. The most effective concentration of earthworm extract was 3 mg/ml, significantly increasing osteoblast proliferation and differentiation, matrix calcium deposition and the expression levels of alkaline phosphatase, osteopontin and osteocalcin. Conversely, 3 mg/ml earthworm extract significantly reduced the tartrate-resistant acid phosphatase activity of osteoclasts without altering cell viability. CONCLUSIONS:Earthworm extract has beneficial effects on bone cell cultures, indicating that earthworm extract is a potential agent for use in bone regeneration.
Study of analgesic effect of earthworm extract.
Luo Wei,Deng Zhen-Han,Li Rui,Cheng Guo,Kotian Ronak Naveenchandra,Li Yu-Sheng,Li Wen-Ping
Pain represents a major clinical problem and one which has exercised generations of healthcare professionals. Earthworms are used as a traditional Chinese medicine, and have been applied pharmacologically and clinically since a long time in China. However, the analgesic effects of earthworm extract (EE) are seldom studied. Hence, we evaluated the analgesic effects of EE in mice. The obtained data showed that EE increased pain threshold and exhibited peripheral but not central analgesic effects in mice; evidenced by increased inhibition ratio in acetic acid writhing test and formalin test, whereas only slight increase in inhibition ratio in hot plate test and tail immersion test. In addition, EE decreased serum norepinephrine (NE), 5-hydroxytryptamine (5-HT), and nitric oxide (NO) synthase (NOS) concentration, similar to other analgesic drugs like morphine and aspirin. In a nutshell, the obtained data have demonstrated that EE has peripheral analgesic properties and could be used as a promising analgesic drug.
The effect of earthworm extract on promoting skin wound healing.
Deng Zhen-Han,Yin Jian-Jian,Luo Wei,Kotian Ronak Naveenchandra,Gao Shan-Shan,Yi Zi-Qing,Xiao Wen-Feng,Li Wen-Ping,Li Yu-Sheng
Chronic nonhealing wounds pose a significant challenge to healthcare system because of its tremendous utilization of resources and time to heal. It has a well-deserved reputation for reducing the quality of life for those affected and represent a substantial economic burden to the healthcare system overall. Earthworms are used as a traditional Chinese medicine, and have been applied pharmacologically and clinically since a long time in China. However, there is paucity in data regarding its wound healing effects. Therefore, we investigated the effect of earthworm extract (EE) on skin wound healing process. The obtained data showed that EE has healing effects on local wound of mice. It decreased the wound healing time and reduced the ill-effects of inflammation as determined by macroscopic, histopathologic, hematologic, and immunohistochemistry parameters. The potential mechanism could be accelerated hydroxyproline and transforming growth factor-β secretion-thus increasing the synthesis of collagen, promoting blood capillary, and fibroblast proliferation. It could accelerate the removal of necrotic tissue and foreign bodies by speeding up the generation of interleukin-6, white blood cells, and platelets. It thus enhances immunity, reduces the risk of infection, and promotes wound healing. All in all, the obtained data demonstrated that EE improves quality of healing and could be used as a propitious wound healing agent.
[Protective effect of earthworm active ingredients against endoplasmic reticulum stress induced L-02 hepatocyte apoptosis].
Duan Leng-Xin,Gao Yang,Wu Xin-Fang,Qiu Ke-Ke,Wang Jian-Gang
Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica
This study focused on the protective effect of earthworm active ingredients (EWAs) on hepatocyte apoptosis induced by endoplasmic reticulum stress (ERS) in L-02 cells. The L-02 cells were cultured in vitro. The cell viability was measured with CCK-8, the apoptosis of L-02 cells was detected by flow cytometry, and the relevant protein and mRNA expressions were detected by Western blot and qPCR. According to the findings, tunicamycin (TM) could obviously reduce the survival rate of L-02 cells in a time-dependent and dose-dependent manner. Compared with normal group, the apoptosis rate in model group was significantly increased (<0.05 or <0.01). The protein and mRNA expressions of ERS-related signal molecules, such as GRP78, PERK, eLF2α, ATF4, CHOP and Bax, were significantly up-regulated (<0.05 or <0.01), while Bcl-2 was significantly down-regulated (<0.05 or <0.01). After the administration with different concentrations of EWAs, compared with model group, EWAs could significantly increase the survival rate ofL-02 hepatocyte and decrease the cell apoptosis rates. It could also reduce the protein and mRNA expressions of ERS-related signal molecules, such as GRP78, PERK, eLF2α, ATF4, CHOP and Bax, in a dose-dependent manner (<0.05 or <0.01) and increased the protein and mRNA expressions of Bcl-2(<0.05 or <0.01). These results showed that EWAs had a significantly protective effect on hepatocyte apoptosis induced by ERS in L-02 cells. Its mechanism may be related to the down-regulation of mRNA and protein expressions of GRP78, PERK, ATF4, eLF2α, CHOP and Bax, and the up-regulation, the relief of ERS and the promotion of the proliferation of impaired L-02 cells.
Effect of earthworm active protein on fibroblast proliferation and its mechanism.
Song Shuliang,Wang Yuling,Ji Kai,Liang Hao,Ji Aiguo
CONTEXT:Earthworms have been used as a traditional medicine in China from thousands of years. In recent years, research has demonstrated that earthworm extracts might promote wound healing; however, its mechanism is still unknown. OBJECTIVE:The study investigates the mechanism and effects of earthworm active protein (EAP), on mouse embryonic fibroblast (NIH/3T3) proliferation. MATERIALS AND METHODS:The effects of earthworm active protein (EAP) in different concentrations (0, 25, 50, 100, 150, and 200 μg/mL) on NIH3T3 cell were detected by the MTT and Brdu incorporation assay (50, 100, and 150 μg/mL). The effects of EAP (37.5, 75, and 150 μg/mL) on the cell cycle were detected by flow cytometry. The cell signaling pathways of EAP-promoting NIH3T3 cell proliferation were studied by the MTT and Western blot by using different signaling pathway inhibitors. RESULTS:The results showed that EAP (50, 100, and 150 μg/mL) could promote NIH3T3 fibroblasts proliferation (36.4 ± 4.4%, 59.1 ± 4.9%, and 71.5 ± 5.7%). The mechanism of EAP promoting NIH3T3 cell proliferation should be as follows: EAP elevated cyclin D1 expression by activating MEK/ERK signaling pathway, and then promoted cell cycle from G1 to S phase, finally caused the proliferation of NIH3T3 cell. PI3K signaling pathway may be the upstream of MEK/ERK signaling pathway. DISCUSSION AND CONCLUSION:The study demonstrates that EAP is effective in promoting effects on proliferation and migration activity of NIH3T3 cell, and the proliferation activity of EAP on NIH3T3 cell may be achieved through the PI3K→Rac→PAK→MEK signaling pathway.
The effect of earthworm extract on mice S180 tumor growth and apoptosis.
Deng Zhenhan,Gao Shanshan,Xiao Xiang,Yin Ni,Ma Shiyang,Li Wenping,Li Yusheng
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Great efforts have been made to explore the potential treatment for cancers, and the most common therapies include surgery, chemotherapy and radiotherapy. As an alternative medication, earthworms have drawn increased attention considering its abundance in resource, easy access and minor side effects compared to traditional therapies. However, few studies had focused on the antitumor effect of earthworm-derived components. The purpose of this study is to investigate whether earthworm extract has an effect on tumor cell apoptosis and growth. Earthworm extract (EE) was purified through multiple steps of centrifugation and chromatography. Mice were inoculated with ascitic fluid derived from mice inoculated with S180 sarcoma tumor cells and fed orally with different amounts of EE for 25 days. Tumor samples were analyzed for size and cell apoptosis. And we found that the weight and sizes of tumor decreased gradually as the amount of EE administered increased. More apoptotic cells and lowered level of lactate dehydrogenase (LDH), a biomarker of tumor invasiveness, was detected in EE-treated group than the untreated group. Our results suggested that EE could dramatically promote tumor apoptosis and reduce tumor size in vivo, suggesting a novel alternative therapy for cancer patients.
Conserved lamin A protein expression in differentiated cells in the earthworm Eudrilus eugeniae.
Kalidas Ramamoorthy M,Raja Subramanian Elaiya,Mydeen Sheik Abdul Kader Nagoor Meeran,Samuel Selvan Christyraj Johnson Retnaraj,Durairaj Selvan Christyraj Jackson,Nino Gopi D,Palanichelvam Karuppaiah,Vaithi Arumugaswami,Sudhakar Sivasubramaniam
Cell biology international
Lamin A is an intermediate filament protein found in most of the differentiated vertebrate cells but absent in stem cells. It shapes the skeletal frame structure beneath the inner nuclear membrane of the cell nucleus. As there are few studies of the expression of lamin A in invertebrates, in the present work, we have analyzed the sequence, immunochemical conservation and expression pattern of lamin A protein in the earthworm Eudrilus eugeniae, a model organism for tissue regeneration. The expression of lamin A has been confirmed in E. eugeniae by immunoblot. Its localization in the nuclear membrane has been observed by immunohistochemistry using two different rabbit anti-sera raised against human lamin A peptides, which are located at the C-terminus of the lamin A protein. These two antibodies detected 70 kDa lamin A protein in mice and a single 65 kDa protein in the earthworm. The Oct-4 positive undifferentiated blastemal tissues of regenerating earthworm do not express lamin A, while the Oct-4 negative differentiated cells express lamin A. This pattern was also confirmed in the earthworm prostate gland. The present study is the first evidence for the immunochemical identification of lamin A and Oct-4 in the earthworm. Along with the partial sequence obtained from the earthworm genome, the present results suggest that lamin A protein and its expression pattern is conserved from the earthworm to humans.
Earthworm protease in anti-thrombosis and anti-fibrosis.
Wang Xiu-Mei,Fan Shi-Chao,Chen Yao,Ma Xiao-Feng,He Rong-Qiao
Biochimica et biophysica acta. General subjects
BACKGROUND:Earthworms are widely used in basic and applied research in medicine, food, environment and agriculture, in which for instance earthworm protease has its own biochemical features. SCOPE OF REVIEW:This review summarizes earthworm protease biochemical features in anti-thrombosis and anti-fibrosis, and provides new perspectives for earthworm to be used in biochemical and pharmaceutical studies. MAJOR CONCLUSIONS:Earthworm protease functions in anti-thrombosis by its fibrinolytic activity and inhibiting platelets aggregation, and anti-fibrosis by its decreasing fibronectin, collagen and laminin, showing a broad substrate specificity. The protease regulators (U3EE) from earthworm also has multiple functions acting as an activator and an inhibitor on different target proteins. Nonetheless, the protease improves the substrate selectivity through substrate-induced changes in the protease active site conformation impact on subsequent reactions with substrates. GENERAL SIGNIFICANCE:It is predictable that both biochemical and applied studies of earthworm proteins including protease will be wider and deeper in the future.
High-pressure tolerance of earthworm fibrinolytic and digestive enzymes.
Akazawa Shin-Ichi,Tokuyama Haruka,Sato Shunsuke,Watanabe Toshinori,Shida Yosuke,Ogasawara Wataru
Journal of bioscience and bioengineering
Earthworms contain several digestive and therapeutic enzymes that are beneficial to our health and useful for biomass utilization. Specifically, earthworms contain potent fibrinolytic enzymes called lumbrokinases, which are highly stable even at room temperature and remain active in dried earthworm powder. However, the high-temperature sterilization method leads to the inactivation of enzymes. Therefore, we investigated the effect of high-pressure treatment (HPT) (from 0.1 MPa to 500 MPa at 25°C and 50°C) on the enzymatic activity of lumbrokinase (LK), α-amylase (AMY), endoglucanase (EG), β-glucosidase (BGL), and lipase (LP) of the earthworm Eisenia fetida, Waki strain, and its sterilization ability in producing dietary supplement. LK showed thermo- and high-pressure tolerance. In addition, HPT may have resulted in pressure-induced stabilization and activation of LK. Although AMY activity was maintained up to 400 MPa at 25°C, the apparent activity decreased slightly at 50°C with HPT. EG showed almost the same pattern as AMY. However, it is possible that the effects of temperature and pressure compensated each other under 100 MPa at 50°C. BGL was shown to be a pressure- and temperature-sensitive enzyme, and LP showed a thermo- and high-pressure tolerance. The slight decrease in apparent activity occurred under 200 MPa at both temperatures. Furthermore, the low-temperature and pressure treatment completely sterilized the samples. These results provide a basis for the development of a novel earthworm dietary supplement with fibrinolytic and digestive activity and of high-pressure-tolerant enzymes to be used for biomass pretreatment.
Anticancer Activity of Earthworm Powder (Lumbricus terrestris) Against MCF-7 and PC-3 Cancer Cell Lines.
Shafi Farha A Ali,Faleh Nebrass
Journal of gastrointestinal cancer
BACKGROUND AND AIM:The importance of earthworm in treatment of various diseases has been proven in ancient literatures. Nowadays, with advances in biotechnology, earthworm is considered a rich natural source of many biomolecules that possesses antioxidant and antitumor activities. The present study aimed to evaluate the antitumor activity of earthworm powder (Lumbricus terrestris) against two cell lines, breast cancer cells (MCF-7) and prostate cancer cells (PC-3). METHODS:Fully matured earthworms (L. terrestris) were collected from soil in Baghdad, Iraq. To assess the cytotoxicity of earthworm powder, the MTT assay was conducted on cancerous (MCF-7 and PC-3 cells) and normal cell line (WRL68 cells) lines. RESULTS:It was revealed that earthworm powder exerts cytotoxic effects against two cancer cell lines. The viability of MCF-7 and PC-3 cells decreased with increasing the concentration. The IC50 values for PC-3 and MCF-7 cell lines were 265.5 and 965.9 μg/ml, respectively, while the earthworm powder exhibited no cytotoxicity against the WRL68 cells. According to the analysis of the results of the multiple cytotoxicity assay (HCS), the treatment of PC-3 cells with 100, 200, and 400 μg/ml of earthworm powder for 24 h at 37 °C led to cell death by changing the permeability of mitochondrial membrane resulting in cytochrome C release and inducing apoptosis. CONCLUSION:The results of the present study contribute additional evidence for the antitumor activity of earthworm extracts. Therefore, further research should concentrate on isolating and identifying the earthworm's active biomolecules that have antitumor activity by investigating the molecular mechanism, genetics, and pathways responsible for the antitumor activity of these biomolecules.
Differential Engagement of Fermentative Taxa in Gut Contents of the Earthworm Lumbricus terrestris.
Meier Anja B,Hunger Sindy,Drake Harold L
Applied and environmental microbiology
The earthworm gut is an anoxic, saccharide-rich microzone in aerated soils. The apparent degradation of diverse saccharides in the alimentary canal of the model earthworm is concomitant with the production of diverse organic acids, indicating that fermentation is an ongoing process in the earthworm gut. However, little is known about how different gut-associated saccharides are fermented. The hypothesis of this investigation was that different gut-associated saccharides differentially stimulate fermentative microorganisms in gut contents of This hypothesis was addressed by (i) assessing the fermentation profiles of anoxic gut content microcosms that were supplemented with gut-associated saccharides and (ii) the concomitant phylogenic analysis of 16S rRNA sequences. Galactose, glucose, maltose, mannose, arabinose, fucose, rhamnose, and xylose stimulated the production of fermentation products, including H, CO, acetate, lactate, propionate, formate, succinate, and ethanol. Fermentation profiles were dependent on the supplemental saccharide (e.g., glucose yielded large amounts of H and ethanol, whereas fucose did not, and maltose yielded large amounts of lactate, whereas mannose did not). Approximately 1,750,000 16S rRNA sequences were affiliated with 37 families, and phylogenic analyses indicated that a respective saccharide stimulated a subset of the diverse phylotypes. An -related phylotype displayed a high relative abundance in all treatments, whereas key -affiliated phylotypes were stimulated by some but not all saccharides. Collectively, these results reinforce the likelihood that (i) different saccharides stimulate different fermentations in gut contents of the earthworm and (ii) facultative aerobes related to and can be important drivers of these fermentations. The feeding habits of earthworms influence the turnover of elements in the terrestrial biosphere. The alimentary tract of the earthworm constitutes an anoxic saccharide-rich microzone in aerated soils that offers ingested microbes a unique opportunity for anaerobic growth. The fermentative activity of microbes in the alimentary tract are responsible for the production of (i) organic compounds that can be assimilated by the earthworm and (ii) H that is subject to emission by the earthworm and can be trophically linked to secondary microbial events in soils. To gain insight on how fermentative members of the gut microbiome might respond to the saccharide-rich alimentary canal, this study examines the impact of diverse gut-associated saccharides on the differential activation of fermentative microbes in gut contents of the model earthworm .