NADPH Inhibits Myocardial Ischemia-reperfusion Injury Through Activation Of AMPK/mTOR Pathway

Aim:This study was to investigate the role of reduced nicotinamide adenine dinucleotide phosphate(NADPH)in myocardial ischemia-reperfusion injury and to explore its potential mechanism of myocardial protection.Methods:The rat model of myocardial ischemia-reperfusion injury(I/R)was established by ligation of the left anterior descending branch of the heart.The experimental animals were randomly divided into 6 groups(n=6):sham operation group(Sham group),myocardial ischemia reperfusion group(I/R group),NADPH4mg/kg treatment group(N4 group),NADPH8mg/kg treatment group(N8 group),NADPH 16mg/kg treatment group(N16 group)and Diltiazem treatment group(Diltiazem group).The sham group was the same as the I/R group except that the left anterior descending artery was not ligated.The administration group received 16 mg/kg of NADPH intravenously at the beginning of reperfusion,and the injection was completed within 5 minutes.Myocardial infarct size was measured by comparing red versus white tissues using Evans Blue and TTC(2,3,5-triphenyltetrazolium chloride)double staining.Sham-operated tissue was removed from the presumed risk region.NADH and NADPH levels in tissues and serum were detected using the NADP+/NADPH,NAD+/NADH assay kit.Damage of tissues was detected by the LDH Cytotoxicity assay kit and the troponin T test assay kit.The levels of reactive oxygen species(ROS)in the myocardium was determined with DHE staining.Rat neonatal cardiomyocytes(NRCM)were cultured from the 1-3 days old SD rats.NRCM was incubated with glucose-free balanced salt solution HBSS,then an oxygen-glucose-deprivation/Reperfusion(ODD/R)model was established with 95%N2 and 5%O2.We chose the appropriate concentration and action time of NADPH by testing the survival rate and release of LDH in cells.The experiment was randomly divided into 4 groups:CON group,OGD/R group,low dose administration group and high dose administration.Flow cytometry and Hoechst staining were used to detect the protective effect of NADPH in injured cardiomyocytes.The effect of drugs on the expression of apoptosis-related proteins and mitochondrial damage-associated proteins in myocardial injury was detected by Western blot and changes in AMPK-mTOR signaling pathway proteins were further detected.Results:In vivo,exogenous NADPH could get into myocardial cells.Administration of exogenous NADPH reduced myocardial infarct size,which was most pronounced at 16 mg/kg,and NADPH could also reduce serum LDH and cTnT levels.Ischemia and hypoxia induced mitochondria to produce ROS,and mitochondrial membrane proteins COXIV and TOM20 were decreased.After NADPH administration,ROS production was reduced and COXIV and TOM20 levels were increased.In vitro,NADPH60nM(N(L))pretreatment significantly increased cell viability in NRCM.In the injury of NRCM induced by hypoxia and hypoxia,low dose NADPH can significantly reduce the apoptosis rate of cardiomyocytes,and could also greatly reduced the expression of cleaved PARP and cleaved-caspase3,the same result could be seen in the animal experiment.NADPH could promote the phosphorylation of AMPK,and down-regulate the expression of mTOR.Administration of the AMPK inhibitor Compound C in vitro could inhibit the phosphorylation of AMPK and attenuate the pro-survival effect of NADPH.Conclusion:All these results suggest that exogenous NADPH shows cardioprotection in myocardial ischemia-reperfusion injury through activation of AMPK/mTOR pathway and inhibition of cardiomyocyte apoptosis.NADPH may become a potential candidate for the prevention and treatment of myocardial ischemic diseases.