Research Dioscin Collagen-induced Arthritis In Rats And Its Mechanism

Rheumatoid arthritis (RA) is an autoimmune disease that expresses chronic inflammation in synovial tissues and joints and develops into impaired joint function. The prevalence of RA varies worldwide between0.5%and1%. Genetic, hormonal, and environmental factors contribute to its development. A major obstacle to the development of rational treatment strategies is that the disease mechanisms and the causative environmental and genetic factors remain largely unknown. Clues may come from experimental arthritis.Dioscorea nipponica Makino is a Chinese tradltional herbal medieine(CTM). Effective parts and chemical constituents of Dioscorea nipponica Makino includes dioscin, gracillin, asperin,25-D-spirosta-3,5-diene and piscidic acid. Dioscin was extracted and purified by methods of solvent extraction,column chromatography, layer chromatography, et al.Collagen-induced arthritis (CIA) is a well-known disease model for RA. CIA resembles RA in a number of pathological, histological and immunological aspects. Features of CIA include chronic synovitis,inflammatory cell infiltration, Pannus formation,destruction of cartilage, and bone erosion. Immune mechanisms that include both humoral and cellular immunity to CⅡ have been implicated in the pathogenesis of the disease. Furthermore, the similarities between the joint pathology in CIA and RA are most widely used for studies of RA pathogenesis and for screening of new drugs for treatment of the rheumatoid disease. This study delineates the disease course, the influence of the organ-and tissue-specificity of inflammation, and the dynamics of the joint inflammation including infiltrating cell types, the influence of T cells, the humoral and cellular reactivity to the arthritogenic cartilage autoantigens rat collagen typeⅡ(CⅡ).The goal of this study was to extend our understanding of the roles played by Dioscin in the pathogenesis of arthritic diseases by interfering with cytokine-signaling pathways. Our study also aimed to develop a new therapeutic strategy for the treatment of RA by blockade of intracellular cytokine-signaling pathways.1.Effccts of Dioscin on inflammatory and immune responses of collagen-induecd arthritis in vivoCIA model was induced by native chicken type Ⅱ collagen that had been dissolved overnight at4℃in0.1M acetic acid(4mg·mL-1) and emulsified with an equal volume of Freund’s incomplete adjuvant(FIA). Hindpaw volumes of rats were measured by volume meter(△ml) and arthritic index was evaluated at the same time; weight gain and arthritic index of immune organ(thymus and spleen) were also examined; antibodies to CⅡ was determined by ELISA and the delayed-type hypersensitivity(DTH)(△mm)was also examined; The legs and hindpaws of rats were removed, fixed with10%Paraformaldehyde in PBS, and then decalcified for10days with EDTA and embedded in paraffin for histologic analysis. The paraffin sections were stained with hematoxylin and eosin;It was shown that the administration of Dioscin (30,60,120mg·kg-1, ig×7d) inhibited the inflammatory response and restored bodyweight and the weight of immune organs of CIA rats. Dioscin had no effect on the concentration of antibodies to CⅡ. It was found that there were increases of lymphocyte proliferation、IL-2production and synoviocytes proliferation in CIA rats, together with IL-1andTNF-a in peritoneal microphages.Dioscin reduced above changes significantly and had good effect on the progressive inflammatory, degeneration of synovial, cartilage, and bone in arthritic joints of CIA rats.2. Effects of Dioscin on immune cells and cytokines of collagen-induced arthritis in vivo and vitroConA and LPS-induced lymphocytes proliferation, the production of interleukin-2(IL-2) secreted by thymus, the production of interleukin-1(IL-1) secreted by peritoneal macrophages(PMφ) were determined by MTT assay; TNF-α level of PMφ were determined by quantitation of the cytotoxic activity of TNF-a on L929cells respectively. Results showed that the increases of lymphocyte proliferation and IL-2production in CIA rats could be inhibited by Dioscin30,60,120mg·kg-1, ig×7din vivo and with0.5,2.5,12.5,62.5,125mg·L-1invtro, the same results as that of IL-1and TNF-α in PMφ.3. Effects of Dioscin on synovial cells and cytokines of collagen-induced arthritis in vivo and vitroSynovial cells were prepared by collagenase and trypsin digestion of small minced membranes. Synoviocytes proliferation and IL-1levels were determined by MTT assay; TNF-α level of synovial cells were determined by quantitation of the cytotoxic activity of TNF-α on L929cells, respectively. Results showed that the increases of synovial cells proliferation in CIA rats could be inhibited by Dioscin at the administration of30,60,120mg·kg-1, igx7d in vivo and at concentration of o.5,2.5,12.5,62.5,125mg·L-1in vitro, the same results as that of IL-1and TNF-a in synovial cells.4. Effects of Dioscin on the expressions of NF-κBp65subunit and COX-2in rats’ synovium of ankle jointThe expressions of NF-κBp65subunit and COX-2in rats’ synovium of ankle joint were determined by Western blot method. we found that NF-κBp65subunit and COX-2expression in CIA rats was more high than that of normal, which could be inhibited by Dioscin at the administration of60,120mg·kg-1igx7d, Results showed that Dioscin could lower the levels of NF-KBp65subunit and COX-2in rats’ synovium of ankle joint.