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Effect of Type Ⅱ Collagen Protein Extracted and Purified from Zaocys on Rat with Adjuvant Induced Arthritis (AA)

Author: PangJie
Tutor: LiJuan
School: Southern Medical University,
Course: Traditional Chinese Medicine
Keywords: Type II collagen Zaocys Adjuvant arthritis Cytokines Synovial cells
CLC: R285
Type: Master's thesis
Year: 2009
Downloads: 156
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Abstract

Background Rheumatoid arthritis (rheumatoid arthritis, RA) in synovial dysplasia pathological features of an autoimmune disease. RA is a refractory disease, either complete remission of the disease, and basic relief, or to reduce the level of disease activity are in need of long-term medication. But has not yet found a drug (drugs including methotrexate, gold preparations for Disease Control), able to completely control the disease. Domestic and foreign researchers tried to control the progression of the disease in various combination regimens, there had been \such as gold, penicillamine, invalid coupled with cytotoxic drugs, and finally with the hormone. But the biggest drawback of this way the use of anti-inflammatory painkillers six months invalid, the bone has been destroyed with disease modifying drugs, can not be restored to the existing joint destruction. Another down the steps mode, that combination first, then gradually less stopped, finally leaving only the two minimal side effects drug maintenance, but what kind of program is the best choice for the long-term effect of the combination therapy , no one can be sure to respond. And either mode of administration can not avoid side effects brought about by the disease control drug non-specific inhibition of the systemic immune system. Biological agents have been started in the country and gradually widely used home and abroad have reported on the rapid suppression of inflammation and improvement of the RA bone destruction, but less because of its long-term efficacy observed to increase the risk of infection of the unexpected, expensive while limiting its clinical application. Mainly in the pathogenesis of RA antigen fuzzy recognition research, its pathogenesis, can inhibit T cell immune responses against self-antigens, recovery the body exposed autoantigen tolerated, beneficial in the treatment of autoimmune diseases. Oral tolerance refers to oral (Oral tolerance, OT) proteins cause autoimmune disease autoantigen, induced activation of immune cells and cytokines, inhibition of in vivo immune responses against self-antigens, to achieve the purpose of the treatment of autoimmune diseases. This safe, effective and convenient treatment method gradually by the attention of scholars at home and abroad. Nearly 20 years, OT has become a hot topic in the immunotherapy. OT of the basic mechanisms involved in active suppression (active suppression), pathway inhibitor (bystander suppression), clonal deletion (clonaldeletion) Cloning incompetence (clonal anergy), the secretion of inflammatory cytokines of the TGF-beta inhibitory and Th1 cytokines, and Th2 cells balance between the factors. Which way to play a dominant role in the oral tolerance depends largely on the dose and type of the oral antigen and own immune state. Low doses of active suppression mediated and bypass inhibition, whereas high-dose-mediated cloning incompetent or missing. In recent years, the oral immune tolerance has become a research focus of the mucosal immune, and is widely used for allergic cerebrospinal meningitis, RA, type I diabetes, autoimmune thyroiditis, experimental autoimmune myasthenia gravis and other autoimmune disease studies. The treatment of autoimmune diseases, clinical and basic experiments reported at home and abroad have about oral antigen. II collagen (type Ⅱ collagen, C Ⅱ) is one of the most abundant protein in the human and animal joints is considered the most likely of RA autoantigen. The various species of collagen with a high degree of homology, about 90% of the same amino acid sequence. At home and abroad both by oral administration of C II induction of immune tolerance treatment of animal models of RA reported that the method is simple, safe and effective, is the focus of current research. The Zaocys is commonly used in traditional Chinese medicine for the treatment of joint pain, principal component analysis Zaocys decoction contains a lot of collagen substances. Adjuvant-induced arthritis (adiuvant arthritis, AA) with people in the clinical manifestations, pathology, immunology, changes and pathological mechanisms RA has many similar characteristics, the more extensive and mature used in RA clinical and basic research . The extract the active ingredients Zaocys, preliminary study and conducted its the AA rats intervention mechanism, help us to understand the mechanism of the treatment of RA Zaocys. The purpose of extraction and purified Zaocys C II 2 through concept Chawu snake C II treatment effects of adjuvant-induced arthritis model, initial exploration of its mechanism of action. Method 1 the Zaocys C Ⅱ extraction and identification of 1.1 to take restrictive pepsin degradation extraction Zaocys C the II; 1.2SDS-PAGE gel electrophoresis determination of protein molecular weight extract of C Ⅱ Zaocys; 1.3 UV spectrophotometer extraction Zaocys snake C Ⅱ UV absorption wavelength; extracted 1.4western-blot identified Zaocys C II; 2 Zaocys C Ⅱ the AA rats intervention effect of 2.1 with complete Freund's adjuvant (complete Freud's adjuvant, CFA) 0.1 ml of foot pad subcutaneous injection induced AA rat model; 2.2 experimental grouping: Ukraine snake C Ⅱ in vitro direct role in the concentration of the program: the blank control group (Control), Ukraine snake C Ⅱ low dose group (low doses, LD), Ukraine snake C Ⅱ dose group (middle doses, MD), Zaocys C II high-dose group (high doses, HD), respectively, for the C II 0μg/ml 1μg/ml, 10μg/ml, 100μg/ml; fed Zaocys C II concentration program: Ukraine the snake C Ⅱ low dose group (low doses, LD), Ukraine snake C Ⅱ in dose group (middle doses, MD), Ukrainian shoot snake C Ⅱ high-dose group (high doses, HD), respectively irrigation service C Ⅱ 5μg/kg 50μg / kg, 500μg/kg; the normal rats AA model group fed with 0.01 mol / L of glacial acetic acid (C Ⅱ solvent); 2.3 with high, medium, and low-dose C II fed SD rats model set blank the control group and the AA model group: modeling after 16 days, 20 days, 24 days, 28 days observation swollen joints, 28 days after modeling separation synovial HE staining observed synovitis; 2.4L929 cytotoxicity assay TNF -α cytotoxic activity, the MTT assay IL-1β Cell proliferation activity; 2.5ELISA assay serum and synovial cell supernatant cytokine level; 3 statistical treatment: results expressed as mean ± SD were using SPSS13.0 software analysis, joint swelling score using repeated measures analysis of variance statistics, analysis of variance was used to compare the one-wayANOVA among groups, homogeneity of variance, LSD test was used to compare the two groups, such as group compared with the control group use Dnutte inspection when for unequal variances using Welch robust estimation, again using T ₂ pairwise comparison, P <0.05 was considered statistically significant results the Zaocys C Ⅱ identification 1.1SDS-PAGE gel black snake C II electrophoretic analysis of molecular weight of approximately 118KD to 120 kD; 1.2 spectrophotometer determination of extracted C II absorption peak is about 230nm; bovine nasal septum C Ⅱ match 1.3western-blot determination of its development with both standard; 2 Zaocys intervention effect of 2.1 C II AA rats using induced by injection of complete Freund's adjuvant foot thick Zhi adjuvant arthritis model, the contralateral joint swelling began to appear to the immune 15d, hind legs, ankle and foot joint redness gradually worsened, presents serious systemic manifestations such as weight loss, hair removal, tail swelling. 16, 20 days after the 2.2 fed Zaocys's C Ⅱ AA degree of joint swelling in rats: modeling groups the degree of joint swelling no significant differences in modeling after 24 days, 28 days, in the low-dose C II animals 2.3 the Zaocys C Ⅱ in vitro model the degree of joint swelling improved (P <0.05); impact on the AA rats synovial cytokine activity: Zaocys C II concentrations in vitro the AA rats synovial cell supernatant TNF -α and IL-1β activity no direct effect; 2.4 Ukraine snake C Ⅱ in vitro AA rat synovial cells and intestinal collection of lymphatic Summary (Peyer's patch, PP) cells Total training system the inflammation factor activity of: Ukrainian snake C Ⅱ concentration in group inhibit synovial cells of AA rats and PP total training system supernatant of TNF-α and IL-1β activity Zaocys C II (P <0.01), high concentration group inhibit AA rats slippery film cells and PP total training system supernatant IL-1β activity (P <0.05); 2.5 fed Zaocys' C Ⅱ impact on the AA rats synovial cytokine activity: AA model group compared with the blank control group, synovial supernatant TNF-α and IL-1β activity was significantly increased (P <0.01); the fed Zaocys C Ⅱ low, middle dose group compared with the AA model group, TNF-α activity decreased (P <0.05) ; in dose group compared with the AA model group, IL-1β activity decreased (P <0.05); the 2.6 gavage Zaocys C Ⅱ impact on the AA rat synovial cell supernatant inflammation factor levels: AA model group and blank compared to the control group, synovial supernatant of TNF-α and IL-1β levels significantly increased (P <0.01); C Ⅱ in Zaocys the role of high-dose group and the AA model group compared inhibited synovial supernatant TNF-α and IL-1β levels (P <0.01); Zaocys the C II low-dose group could significantly inhibit synovial supernatant IL-1β levels (P <0.01); Zaocys C II medium dose group significantly increased synovial supernatant of TGF-β (P <0.01): 2.7 gavage C, Zaocys II AA serum inflammatory factors: AA model group compared with the blank control group, TNF-α concentrations (P <0.01), IL-1β concentration was significantly higher (P <0.01); fed Zaocys C Ⅱ, the high-dose group compared with the AA model group, TNF-α concentrations decreased (P <0.01); gavage Ukrainian snake C Ⅱ each dose group with the AA model group compared to, of IL-1β concentration significantly significantly decreased (P <0.01); fed Ukraine snake C Ⅱ, high-dose group compared with the AA model group, TGF-β concentrations rise ( P <0.01); Conclusion restrictive pepsin degradation extraction Zaocys C Ⅱ, consistent with the SDS-PAGE gel electrophoresis, UV spectrophotometer and western-blot method identified physical and chemical nature of the standard C II, to prove we extract for Zaocys C II. 2 the fed Zaocys C Ⅱ can improve the AA rats joint swelling extent and synovitis Zaocys C II in vitro has no direct effect on synovial cytokine C Ⅱ in vitro effects of black snake the AA rats synovial cells and PP the cells common training system and fed Zaocys C Ⅱ can inhibit the synovial cytokine level and activity, inhibition of TNF-α and IL-1β levels and elevated serum level of TGF-β, suggesting Zaocys C Ⅱ through significant intervention effect of oral tolerance mechanism of AA rats.

Effect of Type Ⅱ Collagen Protein Extracted and Purified from Zaocys on Rat with Adjuvant Induced Arthritis (AA)

Abstract

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