The Role And Mechanism Of Autophagy In Regulation IFN-γ-stimulated Sensitivity Of Vascular Smooth Muscle Cells To FasL-mediated Apoptosis

Objective:To investigate the role of IFN-y combined with FasL on vascular smooth muscle cells and to explore the regulation mechanisms of IFN-y inducing cells sensitive to apoptosis.Method:Primary vascular smooth muscle cells were isolated from thoracic aorta of 4-week old Sprague Dawley rat and cultured in DMEM/F12 medium containing 5% FBS,2ng/ml basic fibroblast growth factor,0.5ng/ml epidermal growth factor. According to the different experimental groups, IFN-y or/and FasL was administered to incubate vascular smooth muscle cells. Viable cell counting, flow cytometry and western blotting analysis were applied to assess the effect of different treatment on vascular smooth muscle cells. Immunofluorescence and western blotting analysis were applied to detect the role of IFN-y inducing autophagy and to explore the relationship between apoptosis of autophagy. Use flow cytometry for detection of membrane Fas, and explore the relationship between autophagy and of Fas trafficking. Construct RNA interference lentivirus vectors for cell infection in order to get FAP-1 knock down cells, and investigate the role of FAP-1 in regulation Fas trafficking. The involvement of autophagy in regulation of the expression of FAP-1 was also explored. Western blotting analysis was applied to investigate the role of Jak2/STAT1 signal pathway in regulation of autophagy in response to IFN-y stimulation.Results:In this experiment, treatment with IFN-y or FasL alone did not induce vascular smooth muscle cell apoptosis. However, the combination of IFN-y and FasL induced cells apoptosis. The expression of FAP-1 was remarkably decreased and the expression of membrane Fas was significant increased after IFN-y treatment. However, when autophagy inhibitor 3-MA was pretreated with cells, there was little change of the expression level of FAP-1 and Fas. Moreover, the expression of membrane Fas was up-regulate when the expression of FAP-1 was knocked down by shRNA. Besides, Jak2/STAT1 signal pathway was activated after IFN-y treatment. And when this pathway was blocked, there was little change of cells after IFN-y treatment.Conclusion:IFN-y combined with FasL induced rat vascular smooth muscle cell apoptosis and the regulation mechanisms was mainly controlled by IFN-y induced autophagy which was dependent on the Jak2/STAT1 signaling pathways. Typically, autophagy was induced after IFN-γ treatment, which could selectively degrade FAP-1. Moreover, the degradation of FAP-1 weakened the binding force with Fas which leaded to Fas trafficking. Cell apoptosis would be initiated when FasL bind to membrane Fas,...