| Diabetic nephropathy(DN)is not only a serious complication of diabetic mellitus(DM),but also a major cause of end-stage renal disease(ESRD.The previous studies have suggested that the appearance of DN proteinuria is related to the thickening of glomerular basement membrane(GBM)and mesangial hyperplasia,but resent pathological studies have confirmed that DN is a kind of podocyte disease.Podocyte injury is visible throughout the course of DN.The key to control the occurrence and development of DN proteinuria is to improve the morphology and function of podocyte.Research purposes:In this study,we focuses on the apoptosis and autophagy of podocytes under high glucose conditions,to investigate the effects of high glucose on podocyte injury and autophagy and the protective effect of Guben huayu tongluo complexing agent on podocytes.In order to provide new prevention and treatment targets for diabetic nephropathy.Research methods:The subjects used conditional immortalized mouse podocyte(MPC5),and the use of Guben huayu tongluo complexing agent contained serum to act on the podocytes.,using normal sugar and high glucose as intervention measures,flow cytometry,mRFP-GFP-LC3 fluorescent double-labeled adenovirus,immunofluorescence,Western Blot,and RT-PCR were used to perform two experiments.Part 1: Effect of high glucose on podocyte injury and protection of Guben huayu tongluo complexing agentUsing normal blood glucose(5mmol/L D-glucose)+ blank serum,high glucose(30mmol/L D-glucose)+ blank serum,and high glucose + drug-containing serum to intervene to culture MPC5 cell lines 24,48,72 hours later,by means of morphological observation and flow cytometry to analyze the effect of high glucose on podocyte injury and the protection effect of Guben huayu tongluo complexing agent.Part 2: Effect of high glucose on autophagy of podocytes and regulation of autophagy by Guben huayu tongluo complexing agentUsing normal blood glucose(5mmol/L D-glucose)+ blank serum,high glucose(30mmol/L D-glucose)+ blank serum,and high glucose + drug-containing serum to intervene to culture MPC5 cell lines 24,48,72 hours later,by means of mRFP-GFP-LC3 fluorescent double-labeled adenovirus transfection and Western Blot detection LC3-II/LC3-I,immunofluorescence binding Western Blot detection of autophagic biomarkers-p62/SQSTM1,Western Blot detection Expression of podocyte autophagy-related proteins(Beclin1)and RT-PCR detection of podocyte autophagy-related genes(LC3,p62,and Beclin1),in order to analyze the effect of high glucose on the autophagy balance of glomerular podocytes and the regulation of autophagy of podocytes by Guben huayu tongluo complexing agent.Result: Part 1:1.Morphological observation of cells:high glucose can lead to abnormal morphological changes of podocytes,and Guben huayu tongluo complexing agent can significantly improve the abnormal morphology of podocytes.2.Flow cytometry(Annexin V-FITC/PI double staining)detection of apoptosis: high glucose can induce apoptosis of podocytes.Guben huayu tongluo complexing agent can reduce the apoptosis of podocytes in high glucose environment.Part 2:1.mRFP-GFP-LC3 fluorescent double-labeled adenovirus transfection:high glucose induces podocyte autophagosomes andautolysosomes were reduced,and Guben huayu tongluo complexing agent could increase the number of autophagosomes and autolysosomes.2.Immunofluorescence detection of p62 levels:high glucose lead to excessive accumulation of P62,and Guben huayu tongluo complexing agent can reduce the accumulation of P62.3.Western Blot detection of LC3,P62 and Beclin1 protein levels:high glucose resulted in decreased expression of autophagy-related proteins LC3 and Beclin1,and increased expression of P62;Guben huayu tongluo complexing agent can increase the expression of autophagy-related proteins LC3 and Beclin1,and reduce the expression of P62.4.RT-qPCR detection of LC3,P62 and Beclin1 mRNA levels:the mRNA expression levels of LC3,P62 and Beclin1 showed the same trend as the Western Blot.Conclusion: Part 1: 1.High glucose can cause podocyte injury and increase podocyte apoptosis;Guben huayu tongluo complexing agent can reduce podocyte damage induced by high glucose,reduce podocyte apoptosis,and protect podocytes.Part 2: Part 2: 1.High glucose can inhibit the autophagic level of podocytes.Guben huayu tongluo complexing agent can improve the high glucose-induced podocyte autophagy.2.Guben huayu tongluo complexing agent can regulate the autophagic level of podocytes through Beclin1 to reduce apoptosis. |
