The Effect Of Autophagy On The Expression Of Cartilage Phenotype In The ATDC5 Cells Under Inflammatory Microenvironment

Part1 The effect of IL-1? on proliferatio n?differential and autophagy of ATDC5Objective To investigate the effects of inflammation environmental by IL- 1 beta(IL-1?) on the cell proliferation,the expression of cartilage phenotype,and the function of the autophagy and apoptosis in the ATDC5 cells.Methods ATDC5 cells were stimulated with different concentrations(0,10,20,30,40, 60ng/ml) of recombinant human IL-1? for 48 h,or with the same concentration(30ng/ml) for different time(0,6,12,24,36,48,72h).Then proliferative ablity were detected by CCK-8 cell proliferation assay,and the m RNA and protein of the related to cartilage and autophagy,were detected by reverse transcription polymerase chain reaction(RT-PCR) and Western blot.Results IL-1? induced cell proliferation observably in a time-dependent pattern.The expression of cartilage index Col2 a and Sox9 in IL-1?-treated ATDC5 cells were decreased,while the expression of the related to autophagy index Beclin-1 and LC3 were increased, which reached its maximum at 24 h.Conclusions Inflammatory is not good for ATDC5 cells expressing cartilage index,while occurrence of autophagy in the early intervention(24h) can promote the expression of cartilage indicators in the ATDC5 cells.Part2 Effect of the coactions between Wnt/?-catenin pathway and NF-?B pathway on ATDC5 cells in inflammationObjective To investigate the role of the Wnt pathway on the expression of cartilage phenotype in the ATDC5 cells in inflammation environmental, analyze the interaction between Wnt pathway and NF-?B pathway, providing a feasible solution for keeping cartilage phenotype in inflammation and clinical treatment of articular cartilage injury.Methods ATDC5 cells were cultured in vitro after stimulation either with different concentrations(0,10,20,30,40,60ng/ml) of recombinant human IL-1? for 48 h,or different concentrations(0,3,6,12,18,24 m M) of Li Cl for 48 h,and create an inflammatory microenvironment with IL-1?,then Li Cl was administered or not,in the 48 h conduct realtime-PCR and Western blot for the related to cartilage marker Collagen 2a,Sox9;Western blot for NF-?B pathway related protein Ikk?,I?B, NF-?B and their phosphorylation levels,as well as Wnt/?-catenin pathway associated protein,?-catenin, GSK-3?and their phosphorylation levels, and c-myc expression.Results in IL-1? treated Group,cartilage index Sox9,Col2 a were reduced but the opposite result treated by Li Cl. Western blot suggested that IL-1?+Li Cl group contrast with only IL-1? group had high er expression of NF-?B pathway related protein and higher GSK-3? and its phosphorylation level, higher ?-catenin level and lower ?-catenin phosphorylation level.Conclusion IL-1? induced inflammatory microenvironment was unfavorable for ATDC5 cells cartilage phenotype expression.Li Cl actived GSK-3?,inhibiting ?-catenin phosphorylation,then activation of Wnt pathways and at the same time inhibited NF-?B nuclear receptors I?B expression,thus contributing to ATDC5 cells cartilage phenotype in inflammatory environment.