Protective Effect Of Puerarin On HGHL Induced Injury Of H9C2 Cardiomyocytes And Its Mechanism

Objective: to establish an in vitro model of diabetic myocardial injury induced by high glucose and high lipids(HGHL)in H9c2 cardiomyocytes,and to observe the expression of autophagy related proteins before and after puerarin(PUE)intervention,so as to explore the protective effect and mechanism of Puerarin on the injury induced by HGHL in H9c2 cardiomyocytes.Methods: H9c2 cardiomyocyte injury model was established by HGHL treatment.HE staining and CCK-8 method were used to determine the best intervention time and concentration of HGHL and puerarin.Drug intervention was divided into normal group,model group,chloroquine group,rapamycin group,puerarin group.Model group,chloroquine group,rapamycin group and puerarin group were made by hghl(Hg 33 mmol / L + HL 500 μ mol / L).Chloroquine group,rapamycin group and puerarin group were respectively given chloroquine(25 μ mol / L),rapamycin(0.5 μmol / L),pue(1 mmol / L)/50)In the normal group,DMEM medium of equal volume was added,and the model was made at the same time with drug intervention.The expression of LC3-II,p62,beclin-1 and cleaved caspase-3 were detected by WB,beclin-1 and p62 by immunocytochemistry,LDH release rate and mortality were detected by LDH.Result:1.HE staining results: on the basis of HG 33 mmol / L,H9C2 cells were treated with HL at the concentrations of 250,500 and 1000 μ mol / L,respectively,with time gradients of 12 h,24h,36 h and 48 h,showing that the cell area reached the maximum at 36 h after 500 μ mol / L treatment,and the pathological characteristics of hypertrophy appeared,so as to determine the concentration and time of modeling.2.CCK-8 results: compared with the model group,Puerarin Treatment with 0.5-5mmol / L concentration for 36 hours could improve the cell proliferation activity(P <0.05);when the concentration of Puerarin intervention was 1 mmol / L,the proliferation activity was the highest(P < 0.05),and then the cell proliferation activity began to decline.3.Western blot results: compared with the control group,the expression level of cleaved caspase-3 in the model group increased significantly(P < 0.01);compared with the model group,the expression level of cleaved caspase-3 in the puerarin group decreased(P < 0.05).4.LDH results: compared with normal group,LDH and cell death rate of model group and chloroquine group increased significantly(P < 0.05).Compared with the model group,the LDH release rate and cell death rate of rapamycin group and puerarin group decreased(P < 0.05).5.Western Results: compared with the normal group,lc3 ii and beclin-1 in the model group decreased significantly(P < 0.05),while p62 protein level increased significantly(P < 0.05);compared with the model group,lc3 ii and beclin-1 protein expression in the chloroquine group decreased significantly(P < 0.05),and p62 protein level increased significantly(P < 0.05);l C3-II and beclin-1 protein in the rapamycin group and puerarin group increased significantly(P < 0.05),while p62 protein level decreased significantly(P<0.05)。6.Results of cellular immunohistochemistry: beclin-1 in model group was significantly decreased(P < 0.05),while p62 protein level was significantly increased(P < 0.05);beclin-1 protein in puerarin group was significantly increased(P < 0.05),while p62 protein level was significantly decreased(P < 0.05)Conclusion: Puerarin has an inhibitory effect on the hypertrophy of H9C2 cardiomyocytes induced by HGHL,and its mechanism may be related to the up regulation of l C3-II and beclin-1 protein,the down regulation of p62 protein expression and the reduction of LDH release.