Studies On The Therapeutic Action And Mechanism Of TCDCA In Adjuvant-induced Arthritis Rats

Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with multiple inflammatory mediators that lead to joint damage, synovial inflammation and cartilage and bone damage, which leads to serious dysfunction. Although the etiology and pathogenesis of RA have not been completely cleared, numerous studies have confirmed that immune function disorder, Synovial tissue of joint hyperblastosis, and articular cartilage and bone damage are key pathologic processes in RA development. Although there are a few anti-rheumatic drugs showing effectiveness on treating RA, their side effects and toxicity call for new and more effective natural drugs.Taurochenodeoxycholic Acid (TCDCA) is one of main bioactive substances of bile from animal bile and our laboratory successfully obtain simple substance with high purity from chicken bile acid. In previous stuties, TCDCA showed remarkably inhibition on both acute and chronic inflammation and confered good regulation in humoral immunity, cytoimmunity and phagocytosis of macrophages. Based on the immunological feature of TCDCA, we examined whether intragastric administration of TCDCA has anti-arthritis effects on adjuvant arthritis model in rat. In order to clarify the mechanism of anti-arthritis action of TCDCA, we also investigated its effects on key cytokines and nuclear factor-κB (NF-κB) in RA development progress.This sutdies included five aspects as followed:(1) Induction of AA model and arthritis evalutionCompare to usually inducing methods of AA model, this study sucessfully optimized and screened out a better method, which induced model showed high achievement ratio, stable and typical clinical symptoms. After adopted evaluation criterion of arthritis model, the AA model which induced by the new method, showed followed characteristics:non-injected hind limb ankle and voix pedis became severely red and edematous and its swelling ration was up to25.15%, there were erythema in four limbs and nodules in tail at days15after immunization; there were serious swelling and distension in the four limbs and obvious nodules in tail, arthritis index was up to14.25and non-injected hind limb swelling ration was up to48.09%, X-ray results showed that there appeared unclear dividing line between bones, bone mineral densities, and parenchyma swelling at days30after immunization. Repeated Experiments results showed that the inducing AA model had stable characteristics and its achievement ratio was reached to80%.(2) Therapeutic action of TCDCA on AA model rats.AA rats were administrated with TCDCA (0.1g/kg and0.2g/kg) at days14after immunization, respectively. Evaluted by model evaluation criterion, there were found that the decimated weight of AA rats was remarkably recovered, and the growth rate of was from25.57%up to44.41%and46.02%; The erythema, edematous and nodules was obviously relieveed in four limbs and tail, arthritis index was from13.38down to10.44and10.03, and non-injected hind limb swelling ration was from50.17%down to47.96%and45.83%; X-ray results showed that TCDCA could well ameliorated unclear dividing line between bones, bone mineral densities, and parenchyma swelling. TCDCA confered good anti-adjuvant arthritis activity in rats.(3) Effects of TCDCA on the key cytokines of AA rats in vivo.The contents and mRNA expression of TNF-α、IL-1β、IL-6�'�IL-10were detected by ELISA and RT-PCR in the serum and synovium tissue of AA rats, respectively. These results showed that TCDCA (0.2g/kg.b.w,0.1g/kg.b.w) confered remarkably inhibition in the protein and mRNA expression of TNF-α、IL-1β and IL-6(P<0.05); TCDCA(0.2g/kg.b.w) remarkably increaed the lower content and mRNA expression of IL-10in serum and synovium tissue with continuing treated for31d.(4) Effects of TCDCA on the key cytokines of FLS and PMΦ from AA ratsFLS was cultured by adopted tissue mass cell culture method and obtained FLS showed typical fibroblast-like shape and high purity. The contents and mRNA expression of TNF-α、IL-1β、IL-6and IL-10were detected by ELISA and RT-PCR in the cell supernate and FLS of AA rats, respectively. The concentration of TNF-α, IL-1β and IL-6were markedly decreased by TCDCA (300μg/mL,400μg/mL and500μg/mL) on a concentration-dependent manner. However, there was not markedly influence on IL-10secretion in AA synoviocytes with TCDCA (300μg/mL,400μg/mL and500μg/mL). mRNA expression levels of TNF-α, IL-1β and IL-6were markedly inhibited by TCDCA (300μg/mL,400μg/mL and500μg/mL), meanwhile, TCDCA at concentration of400μg/mL and500μg/mL markedly increased mRNA expression level of IL-10in the synoviocytes of AA ratsPMΦ was obtained by PBS peritoneal lavage mehod. The contents and mRNA expression of TNF-α、IL-1β、IL-6�'�IL-10were detected by ELISA and RT-PCR in the cell supernate and PMφ of AA rats, respectively. The concentration of TNF-a, IL-1β and IL-6were markedly decreased by TCDCA (150μg/mL,180μg/mL and200μg/mL) on a concentration-dependent manner(P<0.05), and TCDCA(150μg/mL and180μg/mL) showed remarkable augment on the concentration of IL-10(P<0.05). Meanwhile, mRNA expression levels of TNF-α, IL-1β and IL-6were markedly inhibited by TCDCA (150ug/mL,180μg/mL and200μg/mL) in PMφ of AA rats.(5) Effects of TCDCA on the NF-κB p65activation and expression in the FLS and PMφ from AA rats.The protein expression of activated NF-κB p65and IκBα was detected by ELISA and Western-blot. It was founded that TCDCA (300μg/mL,400μg/mL and500μg/mL) showed remarkably inhibition in the activated NF-κB p65and phosphorylated IκBα protein expression, and confered augment on the non-phosphorylated IκBα protein in FLS on a concentration-dependent manner; TCDCA (150ug/mL,180μg/mL and200μg/mL) showed remarkably inhibition in the activated NF-κB p65and augment on the non-phosphorylated IκBα protein expression on a concentration-dependent manner, and TCDCA (200μg/mL) remarkably inhibited the phosphorylated IκBα protein expression in PMφ.Conculsions:(1) AA rats with typical and stable clinical symptoms was induced and its achievement ratio was up to80%.(2) TCDCA confered good anti-adjuvant arthritis activity in rats.(3) Anti-arthritis activity of TCDCA could be mediated via that TCDCA directly inhibited the activity of NF-κB p65in FLS and PMφ, and then reduced the protein and mRNA expression of TNF-α、IL-1β and IL-6in AA rats.