Effect Of Regulating PPAR Gamma Expression On Hypoxia-induced Necrotizing Apoptosis In RTEC

Objective:By establishing a model of necrotizing apoptosis in Renal tubular epithelial Cell（RTEC）injured by hypoxia,to silence endogenous or transfect exogenous Peroxisome Proliferators-activated Receptorsγ（PPARγ）by virus transfection technique,so as to regulate its expression level,and to explore the effect of regulating the expression of PPARγreceptor on RTEC necrotic apoptosis induced by hypoxia.Methods:The RTEC cell line（NRK-52E）derived from rat in vitro was cultured and subcultured,and then randomly divided into normal control group,hypoxia injury group,gene overexpression group,and silent group.The normal control group was cultured under normal conditions without any treatment,and the cells in overexpression group and silent group were transfected with exogenous virus or silenced by si RNA,and the transfection rate was more than80%verified by fluorescence microscope and real-time fluorescence quantitative PCR.Then the cells of hypoxia injury group,overexpression group and silent group were put into the hypoxia chamber（perfused with mixed hypoxia gas of950ml/L N₂ and 50ml/L CO₂）for 48 hours to establish RTEC necrotic apoptosis model.After the modeling is completed,taking out and collecting the cells to detect the relevant indexes of the experiment.The morphological changes of four groups of cells before and after hypoxia were observed under light microscope,the cell viability and proliferation were detected by CCK-8（cell counting kit-8）kit;to detect the m RNA expression of cell PPARγ,Receptor Interaction Protein1（RIP）,RIP3,and Transforming Growth Factor-β（TGF-β）by using real-time quantitative Polymerase Chain Reaction（q PCR）;to detect the protein expression of cells PPARγ,RIP1,RIP3,and TGF-βby Western blotting（WB）.Results:1.Under the light microscope,the RTEC cells in the normal control group were flat and elliptical,with full cell bodies,arranged like cobblestone paving,and the intercellular space was moderate.compared with the normal control group,the cells in the hypoxia injury group,overexpression group and silent group showed narrow and long changes,the cytoplasm shrank,the cell density decreased,the cell space widened and the cell fragments increased.Compared with the hypoxia injury group,the cell density in the overexpression group was significantly higher,and the cell space and fragments were significantly decreased;while in the silent group,the cell density decreased significantly,the cell space and fragments increased significantly.2.Compared with the normal control group,the m RNA and protein expression of PPARγin the hypoxia injury group,overexpression group,and silent group were significantly decreased（all P<0.05）;Compared with the hypoxia injury group,the m RNA and protein expression of PPARγin the overexpression group were significantly increased（all P<0.05）,while the m RNA and protein expression of PPARγin the silent group were significantly decreased（all P<0.05）.3.Compared with the normal control group,the m RNA and protein expression of RIP1、RIP3 and TGF-βin the hypoxia injury group,overexpression group and silent group were significantly increased（all P<0.05）;Compared with the hypoxia injury group,the m RNA and protein expression of RIP1、RIP3 and TGF-βin the overexpression group were significantly decreased（all P<0.05）;The m RNA and protein expression of RIP1、RIP3 and TGF-βin the silent group were significantly increased（all P<0.05）.4.Compared with the normal control group,overexpression group and silent group before hypoxia,the activity and proliferation ability of cells in hypoxia injury group,overexpression group and silence group significantly decreased（all P<0.05）.Among the three groups,the cell activity and proliferation ability decreased most significantly in the silent group（all P<0.05）,and the cell activity and proliferation ability in the overexpression group decreased the least compared with the other two groups（all P<0.05）.Conclusion:（1）In hypoxia-induced RTEC necrotic apoptosis,the expression of PPARγis significantly decreased and it participates in RTEC necrotic apoptosis.（2）Up-regulating the expression of PPARγcan reduce the degree of hypoxia-induced RETC necrotic apoptosis.（3）Down-regulation of PPARγexpression can aggravate the degree of RETC necrotic apoptosis induced by hypoxia.