The Effects Of Puerarin On Apoptosis And Autophagy Of RSC96 Cells Induced By High Glucose

Objective:Through high glucose induced damage RSC96 cells establish diabetic peripheral nerve injury model,adopting the effective ingredients of pueraria,puerarin,to intervene,observing the expression of apoptosis and autophagy related proteins before and after puerarin treatment,this paper aims to explore the effects of puerarin on apoptosis and autophagy of RSC96 cells induced by high glucose.Method:RSC96 cell lines were puchased from ATCC cell bank to establish the hyperglycemia injury model of RSC96 cells in vitro.The Cleaved Caspase-3 protein was detected by western Blot?WB?,and the glucose concentration was determined to be 400 mmol.L^-11 after successful modeling.Through the method of CCK-8,it was quite sure that the intervention concentration of puerarin was determined to be 0.5mmol.L^-1,thus they were randomly divided into blank control group,high glucose group,and puerarin intervention group.Blank control group did not add any drugs;high glucose group:after 48h of normal culture,400 mmol.L^-11 of high glucose was added and incubated for 1h;puerarin intervention group:after 48h of normal culture,RSC96 cells were pretreated with 0.5 mmol.L^-11 puerarin,and then 400 mmol.L^-1high glucose was added and incubated for 1h.Finally,cell survival rate was observed by CCK-8 method;expression of Beclin-1 was detected by immunocytochemistry;expression of Cleaved Caspase-3,LC3-II,p62 and Beclin-1 protein was detected by WB.Result:Compared with the high glucose group,the survival rate of RSC96 cells and the expression of p62 protein?P<0.05?were significantly increased?P<0.05?after puerarin pretreatment,and the expression of Cleaved Caspase-3,LC3-II,and Beclin-1 protein was controled?P<0.05?.Details are as follows:1.RSC96 cells were damaged by the treatment with 400 mmol.L^-11 glucose for 6h.After being treated with 400 mmol.L^-11 glucose for 0h,3h,6h,9h,12h and 24h,the expression of proteins in each group was dectected by WB method,the results showed that the expression ofCleaved Caspase-3 protein was significantly increased after being treated with 400 mmol.L^-11 glucose for 6h?P<0.01?.2.Puerarin pretreatment could increase the survival rate of high glucose induced RSC96 cells by?0.1-1?mmol.L^-11 in the concetration range,puerarin was treated for15min.When puerarin intervention concentration was 0.5 mmol.L^-1,the survival rate of RSC 96 cells was higher than the blank control group?P<0.05?.With the increase of puerarin,the cell survival rate decreased.3.Compared with the blank control group,puerarin intervention group reduced the expression of Cleaved Caspase-3 protein.After high glucose treatement,the expression of Cleaved Caspase-3 protein increased significantly?P<0.01?.With puerarin precubation for 15min,the expression of Cleaved Caspase-3 protein was significantly reduced?P<0.05?.4.Compared with the blank control group,the expression of p62 protein in cells was significantly reduced after 400 mmol.L^-11 high glucose for 1h?P<0.05?.Thus we chose 400 mmol.L^-11 of high glucose for 1h when autophagy related proteins were detected.5.Puerarin inhibited the autophagy level of high glucose induced RSC96 cells.Compared with the high glucose group,puerarin intervention group could lower the expression of LC3-II protein?P<0.05?,higher the expression of p62 protein?P<0.05?.The expression of Beclin-1 protein showed the same trend in inmunocytochemistry and WB.Immunocytochemistry showed that Beclin-1 protein was moderate positive in the high glucose group and weakly positive in the puerarin intervention group.WB results showed that the expression of Beclin-1 protein was significantly inhibited in the puerarin intervention group than in the high glucose group?P<0.05?.Conclusion:Puerarin can protect damaged RSC96 cells which are induced by high glucose,and promote proliferation,inhibitate autophagy and apoptosis.It may works as inhibitating the expression of LC3-II protein,Beclin-1 protein,improving the p62 protein.