Research On The Protective Effects And Mechanisms Of TFDG On Inflammation Injury Of Rat Chondrocytes Induced By IL-1?

Osteoarthritis(OA)is the most common degenerative joint disease with morbidity increases obviously while the age rising,which seriously impacting the old people's joint function and life quality.Damage and loss of articular cartilage is the key to pathological process in OA.Inflammatory cytokines such as IL-? can cause cartilage degeneration and inflammatory reaction through a series of cascade reaction,especially in cartilage lesions.It has thousands of years of application history in using plant active ingredients to treat osteoarthritis.Epidemiological studies demonstrate that long-term drinking black tea has protective effects on articular cartilage.Theaflavin-3,3-digallate(TFDG)is the main active ingredient of black tea polyphenols and it has abundant biological activity in inflammatory regulation.The study of the protective effects of TFDG on inflammatory injury in chondrocytes can be regarded as theoretical references for the preventing and curing OA.Therefore,IL-1? is adopted to induce rat chondrocytes that were isolated and cultured in vitro,after which a model of osteoarthritis was built through stimulating rat chondrocytes by IL-1?,thus exploring that the regulating effects and the underlying mechanisms of theaflavin-3,3'-digallate(TFDG)on inflammatory injury of rat chondrocytes induced by IL-1? in this study.The relevant research results are showed as the following.It can be seen from the fluorescent microscope,the staining results of cultured cells presented positive after staining by toluidine blue staining and type ? collagen immunofluorescence staining,which means the cultured cells in the experiment were cartilage cells.It can be found from inverted phase contrast microscopy that IL-1?obviously impaired normal rat chondrocytes morphology.The results from CCK-8 kit,real-time PCR,ELISA,and western blot showed that IL-1?hindered cell viability(P<0.01)and increased inflammatory cytokines IL-1?,IL-6 and COX-2 expression(P<0.001),thus causing inflammatory change.The above data revealed that IL--1?could destroy the cell morphology of chondrocytes and impaired the physiological function of chondrocytes,and this means the inflammatory injury model was successfully built.The CCK-8 assay indicated that 75,100?M TFDG significantly decreased the cell viability of normal rat chondrocytes(P<0.05),but 0?50 ?M TFDG effectively increased the the cell viability of normal rat chondrocytes(P<0.05).Meanwhile,0-50 ?M TFDG blocked the effect of IL-1? on the viability of OA chondrocytes(P<0.01).TFDG could significantly reduce the decline of cell viability induced by IL-1?(P<0.01).It also significantly reduced inflammatory reaction via decreasing pro-inflammatory cytokines IL-1?,IL-6 and COX-2 expression of rat chondrocytes induced by IL-1?(P<0.05).During the anabolism of cartilage matrix,TFDG effectively up-regulated Col II and ACAN mRNA expression(P<0.05),thus demonstrating TFDG promoted cartilage matrix synthesis.During the catabolism of cartilage matrix,TFDG obviously down-regulated MMP-13 and ADAMTS-5 expression(P<0.05),thus proving TFDG inhibited cartilage matrix degradation.Furthermore,the changes in the protein levels of the phosphorylation of JNK and ERK induced by IL-1? were partially inhibited by TFDG in a dose-dependent manner,the changes in the phosphorylation of p38 protein levels were completely inhibited by TFDG,thus indicating a weakening activity of MAPKs.In conclusion,this research first discovered the protective effects and mechanisms of TFDG on inflammatory injury of rat chondrocytes induced by IL-1? The current study suggested that TFDG lessened the release of inflammatory factors in OA chondrocytes,enhanced anabolic activity and inhibited its catabolic activity compared with normal chondrocytes via blocking MAPKs signaling pathway so as to exert the protective effects against OA.The study provides the first evidence that TFDG significantly slow OA disease progression and exert a palliative efffect.