Vitexin Attenuates Myocardial Ischemia/reperfusion Injury In Rats By Regulating Mitochondrial Dysfunction Via Epac1-Rap1 Signaling Pathway

Objective: Vitexin(VT)is a main bioactive flavonoid compound derived from the dried leaf of hawthorn.Recent studies have shown that VT shows cardioprotective effects both in vivo and in vitro,but its protective mechanism has not been fully elucidated.Mitochondrial dysfunction is a salient feature of myocardial ischemia/reperfusion(I/R)injury(MIRI),but the potential mechanism is still unclear.The purpose of this study is to investigate the protective mechanism of VT by regulating Epac1-Rap1 signaling pathway to inhibit mitochondrial dysfunction of ischemic myocardium and reduce cardiomyocyte apoptosis,and to provide new targets and theoretical basis for VT in the treatment of ischemic heart disease.Methods: Isolated SD rat hearts were subjected to MIRI in a Langendorff perfusion system,and H9c2 cells were subjected to hypoxia/reoxygenation(H/R)in vitro.Cardiac hemodynamic changes were detected by biological function systems;myocardial infarction size was detected by TTC staining;myocardial histopathological changes were detected by H&E staining;cardiomyocyte apoptosis was detected by TUNEL staining and Hoechst 33342 staining;reactive oxygen(ROS)levels were detected by DCFH-DA;The change of mitochondrial ultrastructure was observed by transmission electron microscope(TEM);mitochondrial membrane potential(MMP)was detected by JC-1 staining;The expression levels of Epac1,Rap1,Bax,Bcl-2,Cleaved caspase-3/9,NOX4,Drp1,MFN2 and Cyt-c were detected by Western blot.Results:VT alleviated I/R-induced left ventricular dysfunction in isolated hearts,reduced left ventricular infarct size and alleviated myocardial histopathological damage in isolated hearts.In addition,TEM showed that I/R caused outer mitochondrial membrane rupture,cristae disappearance and vacuolation,and VT reduced mitochondrial ultrastructure damage in rats.DCFH-DA results showed that VT significantly reduced the ROS levels of H9c2 cells subjected to H/R;JC-1 staining results showed that VT significantly increased the MMP of H9c2 cells subjected to H/R.TUNEL staining,Hoechst 33342 staining,and Western blotting showed that VT inhibited MIRI-induced cardiomyocyte apoptosis.In addition,Western blot results showed that VT significantly inhibited the expression of Epac1 and Rap1 proteins,and inhibited the recruitment of Bax and the release of Cyt-c in mitochondria of cardiomyocytes.Conclusion: The present study showed that vitexin treatment could reduce MIRI in rats.The underlying mechanism might reduce MIRI-induced mitochondrial dysfunction by regulating the Epac1-Rap1 signaling pathway to maintain mitochondrial dynamic balance,thereby inhibiting the activation of mitochondrial-mediated apoptotic pathways.