Effect Of Type Ⅱ Collagen Protein Extracted And Purified From Zaocys On Rat With Adjuvant Induced Arthritis (AA)

BackgroundRheumatoid arthritis(RA) is an autoimmune disease characterized by hyperplasia of the synovium with resultant cartilage destruction.Although DMARDs has effective therapeutic action on RA,the clinical application of DMARDs will be inhibited because of its' side reaction which nonspecificly suppressed immune system. Biological agents is now widely used,its' rapid inhibition on inflammation and improvement on bone destruction in RA was reported,but because of lack of their long-term efficacy study observed,unexpected increased the risk of infection and which is very expensive,the extensive use of their clinical is limited.As for the study of pathogenesis of RA,a large number of studies focusd on promiscuous recognition. If the reaction of immune response to self-antigen will be inhibited and the tolerance of the body exposed to the self-antigen will be restored,it is conducive to the treatment of autoimmune diseases.To aim directly at the pathogenesis of RA,this new treating method of RA will be paid close attention to.Oral tolerance,a state of immunological unresponsiveness induced by oral administration of antigen,has posed intriguing possibilities for the treatment of autoimmune diseases including RA,multiple sclerosis and insulin dependent diabetes. Oral tolerance has been applied to prevent and treat autoimmune disease in several animal models including arthritis.It is now clear that oral tolerance is an active immunologic process and is mediated by more than one mechanism.Low doses of antigen administration favor the induction of active suppression and bystander suppression,whereas higher doses favor the induction of anergy or deletion.Which way of oral tolerance is dominant role largely depends on the the dose and type of antigen and the immune status of their own.TypeⅡcollagen,which is one of the major components of articular cartilage and provides the tensile strength of this tissue,is a signification autoantigen associated with the development and therapy of RA.TypeⅡcollagen(CⅡ),an orally administered autoantigen in suppressing autoimmune diseases,was first reported in 1986.Since then,a large number of studies have been conducted on the mucosal treatment of arthritis.It is an attractive approach for the treatment of autoimmune and inflammatory diseases thanks to its lack of toxicity,ease of administration over time and its antigen-specific mechanisms of action.Adjuvant induced arthritis have many similar characteristics with RA in the clinical manifestations,pathology,immunology and pathology change mechanism,is currently more widely used in the mature clinical and basic research of RA.Extracted the active ingredients from Zaocys and to study its role on the intervention mechanisms in AA rats,will help us to understand the treatment mechanism of Zaocys on RA.Objective1.Extracted and purified typeⅡcollagen protein from Zaocys.2.To investigate the effect of typeⅡcollagen protein from Zaocys on adjuvant induced arthritis(AA) in rats and explore its' mechanism.Methods1 Extracted and identined typeⅡcollagen protein from Zaocys.1.1TypeⅡcollagen protein was abstracted and purified from Zaocys by Pepsin digestion;1.2Molecular weight of typeⅡcollagen protein from Zaocys detected by SDS-PAGE analys.1.3Ultraviolet absorption of typeⅡcollagen protein from Zaocys detected by ultraviolet spectrophotometer.1.4TypeⅡcollagen from Zaocys protein identined by western-blot. 2 The effect of typeⅡcollagen protein from Zaocys on adjuvant induced arthritis(AA) in rats2.1AA was induced by a single intradermal injection of 0.1ml of Complete Freund's Adjuvant into the right hind paw.2.2Effects of different daily doses of Zaocys CⅡ(0μg/kg,1μg/kg,10μg/kg,100μg/kg,respectively) on TNF-αand IL-1βproduction by synoviocytes of AA rats in vitro was observed.Normal group and AA group was fed with vehicle(0.01 M acetic acid) and different daily doses of Zaocys CⅡ(5μg/kg,50μg/kg,500μg/kg, respectively) wae fed with each treated group2.3After treated with typeⅡcollagen protein,hind paws secondary swelling of rats and inflammation of synovial membrane were observed.2.4Tumor necrosis factor-α(TNF-α) activity was measured by L929 cytotoxicity bioassay and Interleukin(IL-1β) activity was measured by MTT dye reduction.2.5The cytokines level of the synoviocytes' supernatants and the serum were detected by ELISA.3 All data are expressed as means±SD,hind paws secondary swelling was analyzed by Repeated Measures and differences among groups were analyzed by one-way ANOVA followed by LSD test or Dunnett test.A P value of＜0.05 was considered statistically significantResults1 The identification typeⅡcollagen protein from Zaocys.1.1Molecular weight of CⅡtypeⅡcollagen protein from Zaocys is about 118KD～120kD detected by SDS-PAGE analys.1.2The UV absorption peak of typeⅡcollagen protein from Zaocys is about 230nm detected by ultraviolet spectrophotometer.1.3The imaging with the standard line of typeⅡcollagen protein from Zaocys is as same as the bands of the standard CⅡ.2 The effect of typeⅡcollagen protein from Zaocys on adjuvant induced arthritis(AA) in rats 2.1AA was induced by a single intradermal injection of 0.1ml of Complete Freund's Adjuvant into the right hind paw.About 15d after immunization AA began appear swelling of contralateral articulation,swelling of ankle and foot and gradually increase the serious cases,a weight loss,hair removal and other systemic manifestations.2.2Treating with low and middle dose of CⅡsuppressed hind paw secondary swelling measured at day 24 and 28(P＜0.05).2.3Each concentration of CⅡfrom Zaocys had no effect on IL-1βand TNF-αproduction by synoviocytes in vitro.2.4Middle concentration of CⅡsignificantly suppressed the activity of IL-1βand TNF-αproduced by synoviocytes-PP cells co-culture system(P＜0.01).Low and high concentration of CⅡsuppressed the activity of IL-1βproduced by synoviocytes-PP cells co-culture system(P＜0.05).2.5Treating with low and middle dose of CⅡsuppressed the activity of TNF-αproduced by synoviocyte(P＜0.05);Treating with middle dose of CⅡsignificantly suppressed the activity of IL-1 produced by synoviocyte(P＜0.05).2.6Treating with middle and high dose of CⅡdecreased the level of TNF-αin synoviocytes' supernatants(P＜0.01).Treating with low,middle and high dose of CⅡdecreased the level of IL-1βin synoviocytes' supernatants(P＜0.01).Treating with middle dose of CⅡsignificantly increased the level of serum TGF-β(P＜0.01).2.7Significantly decreased the level of serum IL-1βand TNF-αwas observed in middle and high dose of CⅡgroup(P＜0.01).Significantly decreased the level of serum IL-1βwas observed in low dose of CⅡgroup(P＜0.01).Treating with middle and high dose of CⅡincreased the level of serum TGF-β(P＜0.01).Conclusion1 The bands of CⅡsamples extracted by Pepsin digestion and purified from Zaocys could be seen as same as the bands of the standard CⅡ.2 Oral administration of typeⅡcollagen protein from Zaocys suppressed hind paw secondary swelling and inflammation of synovial membrane.CⅡhad no effect on the activity of IL-1βand TNF-αproduction by synoviocytes in vitro,but feeding with CⅡ suppressed the level and activity of cytokine from serum and synovial membrane.Oral administration of typeⅡcollagen protein from Zaocys can effectively treat the AA rats through the mechanism of oral tolerance.