Exprimental Study Of The Tumor Necrosis Factor-like Weak Inducer Of Apoptosis In The Formation Mechanism Of Osteoarthritis

BackgroundOsteoarthritis is one of the most common joint diseases especially in the aged people. The major clinical feature is chronic joint degeneration and globally, the prevalence of OA is the highest compared with other joints diseases. With the increasing of aging of the population, the prevalence of OA is rising as well. The occurrence of OA is contributed to mechanics and biological factors which cause the imbalance between synthesis and degradation of chondrocytes, extracellular matrix and subchondral bone. As the etiology of OA is focused on biomechanical changes for a long time, which singly cannot demonstrate the mechanism of OA comprehensively, more and more studies are transferred to enzymology, inflammatory cytokines and apoptosis and so on.The main pathological process, showed by nowadays’research, is the degeneration of articular cartilage because of the increased expression of various proteases in articular cartilage and synovium, which is due to various pathogenic factors, causing the degradation of network of collagen fibers and proteoglycans in the articular cartilage matrix under the effects of inflammatory cytokines. There are many proinflammatory cytokines and chemokines involved in the regulation of protease and the most important ones are IL-1β and TNF-α which not only play the leading role in inflammatory reaction, but also raise the expression of matrix metalloproteinases(MMPs) and increase its biological activity. As a result, can OA be controlled through the inhibition of IL-10and TNF-α? The clinical test didn’t show the expected results which, in our opinion, is due to other known and unknown factors and the complex interactions between them. The coordination and interaction of them stimulate the synthesis and secretion of MMPs and finally lead to the degradation of the cartilage tissue. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is one of the members of TNF ligand superfamily, named by the activity of inducing the apoptosis of tumor cell. More researches showed that, TWEAK is a multi-functional cytokine expressed in many types of cells. It can lead to cell apoptosis and the secretion of inflammatory cytokines and chemokines, while cartilage formation can be specifically inhibited so as to hinder the endogenous repair of cartilage. Fibroblast growth factor inducible14(Fnl4) is one of TWEAK acceptor. It is wildly expressed in various tissues and can start a series of intracellular signal transduction pathways by connecting different receptor protein or cytosolic protein with TNF receptor-associated factor domain. Does TWEAK also act as a role in the degradation of OA cartilage matrix? Evidence shows that TWEAK is a multifunctional cytokine that is similar to IL-lbeta and TNF-alpha, and whether it also co-participation in the evolution of the articular cartilage degradation? TWEAK and its receptor Fnl4are also play a role in the OA?In our study, rat OA model is established to evaluate the expression of TWEAK, and Fn14mRNA and protein in cartilage and synovial membrane, as well as the influence of TWEAK to the inflammatory cytokines, chemokines, and matrix metalloproteinase produced by vitro cultured cartilage cells and fibroblast-like synovial cells. We intended to have a preliminary understanding of the role of TWEAK and its receptor Fnl4in the pathogenesis of OA and provide theoretical basis for new OA therapy.Part I Establishment and evaluation of rat model of osteoarthritisObjectives To establish rat OA model with gross and histological evaluation of the light microscope for following steps.Materials and methods Anterior cruciate ligament transection is performed for rat model establishment, while sham group and normal group are as control. Gross and histological changes are evaluated in1,2and4weeks postoperatively, with Mankin system score.Results Specific degenerative changes of OA are found by gross and histological observation and with the time passing by, they are getting worse. All the presentations match the OA pathological development process in early and mid-term and facilitated the following molecular biological detection.Conclusions Rat OA model, established by ACLT, is stable and excellent for further research. Part Ⅱ TWEAK and its receptor Fnl4expression in articular cartilage and synovium tissue of rat osteoarthritis.Objectives To evaluate the expression of TWEAK, Fn14mRNA and protein in the knee cartilage and synovium tissue of rat OA model, as well as TWEAK concentration in the synovial fluid.Materials and methods Establish rat OA model with sham group and normal group as control. Cartilage, synovium tissue and synovial fluid is achieved1,2and4weeks postoperatively. Real-time PCR is used to detect the expression of TWEAK and Fn14mRNA; TWEAK and Fn14protein is evaluated by Western-blot while ELISA is used for TWEAK concentration in the synovial fluid.Results The expression of mRNA and protein of TWEAK and Fn14in postoperative2and4weeks is significantly higher than that of sham and normal group in synovial tissue; The expression of mRNA and protein of TWEAK and Fn14in postoperative2weeks is higher than that of sham and normal group in cartilage tissue; The concentration of TWEAK in postoperative1,2,3weeks is higher than that of sham and normal group in cartilage tissue.Conclusions In rat OA model, the expression of mRNA and protein of TWEAK and Fn14is significantly raised in some period during OA development, while the TWEAK concentration in the synovial fluid is elevated as well. This shows the close relationship between them and OA. Part Ⅲ The influence of TWEAK to the production of inflammatory cytokines, chemokines, and matrix metalloproteinase from vitro cultured chondrocytes and fibroblast-like synovial cells.Objectives To evaluated the the influence of TWEAK to the production of inflammatory cytokines, chemokines, and matrix metalloproteinase from vitro cultured chondrocytes and fibroblast-like synovial cells.Materials and methods Normal cartilage and synovium tissues are obtained from Wistar rat. Chondrocytes and fibroblast-like synovial cells are vitro cultured and identified. They are stimulated by TWEAK and liquid supernatant is obtained after24h,48h and72h for matrix metalloproteinase (MMP-1、MMP-3、MMP-9、MMP-13), inflammatory cytokines(IL-1β、IL-6、TNF-α) and chemotactic factor (RANTES、MCP-1、 MIP-2、IL-8) evaluation.Results Under TWEAK stimulation, matrix metalloproteinase (MMP-1, MMP-3, MMP-9), inflammatory cytokines(IL-1β, IL-6, TNF-α) and chemotactic factor (RANTES, MCP-1, IL-8) are significantly elevated, while MIP-2decreased and MMP-13is just increased in chondrocytes. And the extent of the increase associated with the stimulus time is positively correlated while MIP-2is negative. In cartilage cells, the quantity of MCP-1, IL-8, MMP-3and MMP13stay the same as time passed by and so is the quantity of MMP-1and MMP13in fibroblast-like synovial cells.Conclusions TWEAK induced increased production of some matrix metalloproteinase, inflammatory cytokines and chemotactic factors by stimulating the vitro cultured chondrocytes and fibroblast-like synovial cells.