| Objective: Ischemic heart disease((Ischemic heart disease,IHD)is the most common cardiovascular-related disease and the main cause of death in the world.More than 7.5million people die from IHD every year.The reperfusion therapy is the most commonly used and effective method for the treatment of ischemic cardiomyopathy.However,in the process of reperfusion therapy,myocardial injury may be aggravated,that is,the myocardial ischemia-reperfusion injury((myocardial ischemia reperfusion injury,MIRI).Myocardial ischemia-reperfusion injury is one of the most common clinical causes that affect the therapeutic effect and even lead to death in patients with ischemic cardiomyopathy.The occurrence and development mechanism of MIRI is complex,involving a variety of mechanisms.Previous studies have shown that the expression of microRNA(miRNA)changes during myocardial ischemia-reperfusion and participates in regulating the occurrence and development of MIRI,and microRNA(miR)-23 a is an important factor involved in regulating the process of MIRI,but its specific mechanism is not clear.The purpose of this study is to explore the role of miR-23 a in myocardial ischemia-reperfusion injury and its possible mechanism.To sum up,in this study,in order to clarify the role of miR-23 a in myocardial ischemia-reperfusion injury,rat H9c2 cardiomyocytes and isolated rat heart were taken as the research objects,through the establishment of cardiomyocyte hypoxia-reoxygenation(H/R)model and isolated rat heart ischemia-reperfusion(I/R)model.The following two parts were studied:(1)To determine the role of miR-23 a in ischemia-reperfusion injury,the expression of miR-23 a in myocardial ischemia-reperfusion injury was determined by comparing the cardiomyocytes and myocardial tissues of normal group and ischemia-reperfusion injury group,and the effects of over-expression of miR-23 a mimics /inhibitors in hypoxia-reoxygenation injury cardiomyocytes or inhibition of miR-23 a on hypoxia-reoxygenation injury myocardium were observed and compared.(2)We observed and compared the effect of miR-23 a inhibitor on the expression of miR-23 a and the pathway of RISK and SAFE in H9c2 cell,and we also explored the possible mechanism of protection by inhibiting the expression of miR-23 a.Methods:1.Langendorff device was used to establish the model of hypoxia-reoxygenation in isolated rat heart.HE staining was used to observe myocardial injury and TTC staining was used to observe myocardial ischemic necrosis.The hypoxia-reoxygenation model of H9c2 cells was established in a three-gas incubator.Cell Counting Kit-8(CCK-8)was used to detect the survival rate of cardiomyocytes at different time points.RT-qPCR was used to determine the changes of miR-23 a expression in myocardial ischemia-reperfusion injury and hypoxia-reoxygenation cardiomyocytes.2.Hypoxia-reoxygenation injury was performed,48 hours after transfection of miR-23 a mimic(mimic)and inhibitor(inhibitor),in H9c2 cells.The changes of cell activity and apoptosis were detected by CCK-8 and flow cytometry.The changes of myocardial enzymes in cardiomyocytes were detected by LDH and CK-MB ELISA kit.3.miR-23a-sponge and blank control(NC)adenovirus were injected into the tail vein of rats to inhibit the expression of miR-23 a in the myocardium of Wistar rats.3 weeks later,the model of ischemia-reperfusion injury was established by in vitro perfusion.HE staining was used to detect the changes of myocardial tissue,and TTC staining was used to detect myocardial infarction.Myocardial apoptosis was detected by TUNEL staining.4.Double luciferase report was used to verify the targeting relationship between miR-23 a and Rap1 a.H9c2 cells were transfected,and cardiomyocytes transfected with miR-23 a mimic and miR-23 ainhibitor were treated with hypoxia and reoxygenation.The m RNA expression of Rap1 a in H9c2 cells was detected by RT-qPCR.The expression of Rap1 a in H9c2 cells was detected by Western-blot.5.H9c2 cells were transfected with Rap1 a ORF and Vector,and overexpressed Rap1 a,was used to detect the expression of Rap1 a protein by Western-blot.CK-8 was used to detect the changes of cell activity and flow cytometry was used to detect cell apoptosis.Western-blot was used to detect the phosphorylation level of mitochondrial apoptotic protein cleaved-caspase3,cleaved-caspase9 in hypoxia-reoxygenated myocardium and AKT,ERK1/2,STAT3 and JAK2 in anti-apoptotic pathway of cardiomyocytes.6.Western-blot was used to detect the inhibition of miR-23 a expression and the expression of mitochondrial apoptotic protein cleaved-caspase3,cleaved-caspase9 Cyt C in hypoxia-reoxygenation cardiomyocytes.And the changes of phosphorylation level of proteins AKT,ERK1/2,STAT3 and JAK2 in the anti-apoptotic pathway of cardiomyocytes.MPTP detection kit and JC-1 kit were used to detect the effect of inhibition of miR-23 a expression on mitochondrial membrane potential in hypoxia-reoxygenation cardiomyocytes.Results:1.Ischemia-reperfusion injury can increase the expression of miR-23 a in H9c2 cells and myocardial tissue,and the extent of increase is positively correlated with the severity of myocardial injury.2.Compared with the transfected NC group,the overexpression of miR-23 a,in H9c2 cells could aggravate the injury of cardiomyocytes induced by hypoxia-reoxygenation,decrease the cell activity,increase the apoptosis rate and increase the release of myocardial enzymes,on the contrary,inhibiting the expression of miR-23 a could reduce the injury of cardiomyocytes induced by hypoxia-reoxygenation.3.Inhibition of miR-23 a expression in myocardial tissue.Compared with NC group,inhibition of miR-23 a expression could significantly reduce myocardial ischemia-reperfusion injury,myocardial infarction area,myocardial injury and myocardial apoptosis rate.4.Overexpression of miR-23 a in cardiomyocytes can reduce the expression of Rap1 a,while inhibition of the expression of miR-23 a can increase the expression of Rap1 a in cardiomyocytes.5.Compared with the vector group,the overexpression of Rap1 a,in H9c2 cells could reduce myocardial hypoxia-reoxygenation injury,reduce apoptosis,reduce the expression of mitochondrial apoptosis-related proteins,and increase the phosphorylation levels of AKT,JAK2,ERK1/2 and STAT3.6.Inhibit the expression of miR-23 a in H9c2 cells.Compared with the NC group,the expression of apoptosis-related proteins decreased,the phosphorylation levels of AKT,JAK2,ERK1/2 and STAT3 increased,and the decrease of mitochondrial membrane potential was inhibited.Conclusion1.The results showed that the expression of miR-23 a was increased in myocardial ischemia-reperfusion injury,and the high expression of miR-23 a in cardiomyocytes could promote cardiomyocyte apoptosis in myocardial ischemia-reperfusion injury.2.miR-23 a can inhibit the expression of Rap1 a.3.Inhibition of miR-23 a expression can inhibit the activation of mitochondrial apoptosis pathway and alleviate MIRI by activating RISK and SAFE pathway. |
PDF Full Text Request