Study On The Regulation Of Autophagy By Ranolazine Against Hypoxia / Reoxygenation Injury In H9C2 Cells Based On PI3K-Akt-mTOR Signal Pathway

Objective:In the previous study,through drug intervention of autophagy,it was confirmed that the protective effect of ranolazine on ischemia/reperfusion injured cardiomyocytes may be related to the induction of autophagy.On this basis,the molecular mechanism of ranolazine preconditioning against hypoxia/reoxygenation injury of cardiomyocytes based on autophagy and PI3K-Akt pathway was studied.Methods: H9C2 cardiomyocytes in logarithmic phase were given hypoxia/reoxygenation injury model(hypoxia for 4 hours,reoxygenation for 3 hours)to screen the best drug concentration of ranolazine to protect H9C2 cells from hypoxia / reoxygenation injury,and to determine the best concentration of ranolazine.H9C2 cardiomyocytes were randomly divided into normal control group(C),hypoxia / reoxygenation model group(H/R),ranolazine preconditioning group(R),ranolazine + Warman penicillin group(RW),rapamycin group(PA).H9C2 cells in group C were cultured normally,and H9C2 cells in the drug pretreatment group,30 min was treated with drugs and then treated with hypoxia/ reoxygenation.The cell activity and the release of LDH in culture medium were detected by CCK-8 method and lactate dehydrogenase kit,and the rate of LC3 immunofluorescence positive cells was detected by laser confocal microscope.The expression of LC3 II/I,Beclin-1,PI3 K,Akt,p-Akt,m TOR and p-m TOR protein was analyzed by Western blotting.Results: Compared with the Control group,there were a large number of suspended cardiomyocytes in the culture medium of H/R group,and the cell activity decreased by 42%,and the release of LDH increased significantly(P<0.001),and the rate of LC3 immunofluorescence positive cells increased(P<0.05).Compared with Hexandrazin group,20μmol/L ranolazine group and PA group,the myocardial cell activity was significantly increased and LDH release was significantly decreased(P<0.001);There were more cardiomyocytes floating in the culture medium,low cell viability and higher LDH release in the RW group,which had no statistical significance compared with the Hypoxia / reoxygenation model group(P>0.05).Western blotting analysis showed that compared with Control group,the expression of LC3 II/I and Beclin-1 increased significantly(P<0.05),the ratio of p-m TOR/ m TOR decreased(P<0.001),the expression of PI3 K increased,the level of Akt phosphorylation increased,and the ratio of p-Akt/Akt increased significantly(P<0.001).Compared with the Hexandrazin group,20μmol/L ranolazine group and PA group,the expression of LC3 II/I and Beclin-1 was significantly up-regulated,the ratio of p-m TOR/m TOR was decreased(P<0.001),and the ratio of protein-p-Akt/Akt was significantly increased(P<0.001).There was no significant difference in the expression level of each protein between RW group and H/R group(P >0.05).Conclusion:1.Hypoxia / reoxygenation could induce autophagy of H9C2 cardiomyocytes.2.The best concentration of ranolazine to protect H9C2 cells from hypoxia/ reoxygenation injury is 20μmol / L.3.Activation of PI3K-Akt signal pathway,inhibition of m TOR phosphorylation and induction of moderate autophagy in hypoxia/reoxygenation H9C2 cells may be one of the mechanisms of ranolazine in protecting cardiomyocytes from hypoxia/reoxygenation injury.