Insulin Protects Cardiomyocyte Against Hydrogen Peroxide-induced Injury Through Upregulation Of MicroRNA-210

Background:The incidence of acute myocardial infarction is increasing year byyear. Acute myocardial infarction has become one of the diseases with thehighest mortality. During the process of myocardial infarction, hypoxiacan cause cell necrosis and apoptosis. The central of infarct area isgenerally based on myocardial necrosis, and myocardial apoptosis isaround the central area. Coronary artery revascularization treatment suchas stenting can quickly open the occluded coronary artery and avoidfurther expansion of myocardial necrosis. However myocardial ischemiareperfusion injury will also lead to myocardial damage including necrosisand apoptosis. Necrosis and apoptosis of myocardial cells will increaserisks of heart failure. How to reduce the apoptosis of myocardial cells inischemia and hypoxia conditions becomes a hot topic in the research ofmyocardial infarction. Look for ways to block cardiomyocyte apoptosiswill help to prevent and improve heart function. Recently scientistsreported that microRNA-210has anti apoptotic effects in acutemyocardial infarction and ischemia reperfusion. It is expected to becometherapy for acute myocardial infarction, but the specific role andmechanism of micro RNA-210have not been clarified.Oxidative stress plays an important role in the process of myocardialcell necrosis and apoptosis in myocardial ischemia reperfusion. Excessoxygen free radicals break balance of the oxidative and anti-oxidativesystem in oxidative stress. Free radical is formed by reactive oxygen and nitrogen free radicals, it can damage the fat, protein and nucleotide.Reactive oxygen free radical, nitrogen free radicals and hydrogenperoxide plays an important role in myocardial ischemia/reperfusioninjury in the heart. H2O2can be generated in the process of ischemia andreperfusion, and supply oxygen free radicals in oxidative stress injury.Mechanism of H2O2in myocardial oxidative stress injury has not beenelucidated, especially the role of microRNA is not clear.Research shows that Akt is the upstream signal pathway. And insulinis the classic PI3K-AKT agonists, has the effect of anti-oxidative stress.So we propose the hypothesis that insulin can activate PI3K-AKTsignaling pathway, leading to the increase of miRNA-210, whilemiRNA-210has anti apoptotic effects, and then play the role ofanti-oxidative stress and anti-apoptosis. This study is to test thehypothesis by real-time quantitative PCR, MTT, Western Blot, flowcytometry and other methods through cytological experiments.Method:Rat myocardial H9c2cell line was used for the followingexperiments:(1) H9c2cells were treated by different concentrations of hydrogenperoxide, myocardial cell survival rate and apoptosis rate were tested byMTT method and flow cytometry method.(2) H9c2cells were pretreated by insulin and then treated byhydrogen peroxide, myocardial cell survival rate and apoptosis rate ofH9c2cells were tested by MTT method and flow cytometry method.(3) H9c2cells were treated by different concentrations of hydrogenperoxide, then the relative amount of miRNA-210expression was tested byreal time quantitative PCR method in H9c2cells; H9c2cells were treatedby the same concentration for different time, then the relative amount of miRNA-210expression was tested by real time quantitative PCR methodin H9c2cells;(4) H9c2cells were treated by different concentrations of insulin, thenthe relative amount of miRNA-210expression was tested by real timequantitative PCR method in H9c2cells; H9c2cells were treated by thesame concentrations of insulin for different time, then the relative amountof miRNA-210expression were tested by real time quantitative PCRmethod in H9c2cells.(5) H9c2cells were pretreated by insulin and then treated by differentconcentrations of hydrogen peroxide for different time, and the relativeamount of miRNA-210expression was tested by real time quantitativePCR method in H9c2cells.(6) Replication-deficient lentivirus encoding rat miRNA-210precursor (pre-210), miRNA-210inhibitor sponge (anti-210), andmiRNA-scramble (miRNA-scr) were used to construct cell models of overexpression of miRNA-210and low expression of miRNA-210.(7) H9c2cell models of over expression of miRNA-210and lowexpression of miRNA-210were treated by hydrogen peroxide, then cellapoptosis rate were detected by flow cytometry method.(8) H9c2cell models of low expression of miRNA-210werepretreated by insulin and then treated by hydrogen peroxide, myocardialcell apoptosis rate were detected by flow cytometry method.(9) Western Blot method was used to test ratio of phosphorylated Akt/and total Akt expression in H9c2cells treated by insulin and/or hydrogenperoxide.(10) H9c2cells were pretreated by PI3K/Akt inhibitor LY294002, andthen treated by insulin and/or hydrogen peroxide, the relative amount ofmiRNA-210expression was tested by real time quantitative PCR method in H9c2cells.Results:1. The percentage of viable cells were significantly and reverselyassociated with H2O2concentration;2. Insulin has a protective effect against H9c2cell apoptosis inducedby hydrogen peroxide;3. Treatments with hydrogen peroxide, insulin or insulin+hydrogenperoxide were associated with a significant increase in miRNA-210levels;4. Manipulation of miRNA-210expression by gene transfectionshowed that miRNA-210could attenuate H2O2-induced cellular injury.Low expression of microRNA-210can reduce the protective effect ofinsulin;5. Inhibition of the PI3K/Akt pathway by the Akt inhibitorLY294002was associated with a decrease in miRNA-210expression.Conclusion:Insulin stimulated the expression of miRNA-210through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injurythat had been induced by H2O2/oxygen species. Our results provide novelevidence regarding the mechanism underlying the protective effect ofinsulin.