Icariin Attenuated The TNF-?Induced Chondrocyte Apoptosis And Autophagy Inhibition Through Down Regulating NF-?B Signal Pathway

Objective The process of autophagy and apoptosis of chondrocytes is crucial in osteoarthritis(OA)pathogenesis.Researches have elucidated the effects of icariin on anti-inflammation and inhibition of cartilage degradation.Tumor necrosis factor-?(TNF-?)is among the most important inflammatory factors in OA pathogenesis.TNF-? could induce chondrocyte apoptosis and damage through activating NF-?B signal pathway.However,the involvements between the icariin and NF-?B signal pathway in the pathogenesis remains unclear.This research was thus designed and performed to elucidate the conjunctive mechanism of icariin and NF-?B signal pathway on chondrocyte autophagy and apoptosis in vitro.Methods Chondrocytes were harvested and cultured from knee cartilage of SD rats.Different concentration of icariin(0,3,5,7,and 20 ?M)were used to treat the chondrocytes to test the cytotoxicity of icariin.Chondrocytes were divided into 4 groups based on the treated methods: PBS(control group),TNF-?,icariin+ TNF-?,PDTC+ TNF-?.The enzyme?linked immunosorbent assay(ELISA)technique was applied to test the expression levels of inflammatory cytokines as IL-1,IL-6 and IL-12.Proteins were extracted from the cells,separated by SDS-PAGE and transferred to the PVDF membrane.And then the cells were incubated with primary and secondary antibodies.We used the Western blot and RT-q-PCR techniques to test the expression levels of ATG5,ATG7,Bax,Bcl-2 and GAPDH.Flow Cytometry was used to analyze the arrested cell cycle.Apoptotic cell numbers were calculated after chondrocyte staining.The inflammatory productions NO,ROS and Caspase-3/9 were also tested.Immunocytochemical Staining was used to test thep65 expression level in nucleus.Data was gathered for statistical analysis and p<0.05 was set to determine the statistical significance.Results The results of ELISA analysis indicated that compared with the control group,icariin +TNF-? group and PDTC+ TNF-? group,the TNF-? group showed significantly higher expression of IL-1,IL-6,IL-12 and Caspase-3/9(p<0.05).Western blot and PCR results indicated higher expression of ATG5,ATG7,Bcl-2 and lower expression of Bax in the TNF-? group,compared with other three groups(p<0.05).The cell cycle was arrested mostly at G1 phase by TNF-?,while cell cycle treated by icariin or PDTC was arrested at S-G2/M phase.In the TNF-? group,the inflammatory productions NO,ROS and metabolic productions MMP3/9 were significantly higher than other groups(p < 0.05).The expression level of NF-?B p-p65 was significantly higher and I?B? expression level lower in the TNF-? group,compared with other groups(p<0.05).Conclusion The present research suggested that TNF-? could induce the production of various inflammatory factors,activation of chondrocyte degradation and apoptosis,inhibition of autophagy.Icariin could partially reverse the negative effect of TNF-? through activating NF-?B signal pathway.