The Protective Role Of Isoquercetin In Alleviating Hypoxia/reoxygenation Injury-induced Apoptosis Of Cardiomyocytes And The Underlying Mechanisms

Coronary heart disease(CHD)is one of the main causes of human mortality,and owing to the improved living standard of Chinese people,the incidence of CHDin Chinais also increasing year by year.Acute myocardial infarction(AMI)is the most serious manifestation of CHD.Restoration of blood flow(reperfusion)in coronary arteriesafter myocardial ischemia remains the most effective method against AMI.But this method often causes ischemia/reperfusion(I/R)injury because reperfusion itself also triggers the activation of immune responsesand abundant apoptosis of cardiomyocytes,which furthercauses the expansion of infarct size.Oxygen radicals,calcium overload,interplays between vascular endothelial cells and neutrophils might be implicated in this process.Therefore,reducing I/R injury is critical to improve the clinical efficacyagainst AMI,and relevant studies prevail at present.Isoquercitrin is a polyphenolic antioxidant with limited toxicity,which is distributed in the flowers,leaves and fruits of many plants.Isoquercitrinexerts potential therapeutic effects in reducing blood pressure,alleviating capillary fragility,enhancing capillary resistance,expanding coronary artery,increasing coronary blood flow and reducing hyperlipidemia.These pharmacological effects of isoquercitrin are also beneficial for patients with CHD or hypertension.Isoquercitrin shows potential therapeutic effects in the treatment of cardiovasculardiseases,but its role inmyocardial I/R injury and the underlying mechanisms have been rarely reported.Accordingly in the present study,we established cellular and animal models of myocardial I/R injury to determine the protective role of isoquercitrin and the potential mechanisms.Aim:Through in vitro and in vivo models of myocardial I/R injury,the present study aimed to determine the protective role of isoquercitrin against apoptosis of cardiomyocytesand the potential mechanisms.Methods:(1)To establish the cellular model of H/R-injured H9C2 cells and myocardial I/R injured rat model to mimic myocardial I/R injury in vitro and in vivo.(2)To treat the H/R-injured H9C2 cells with different doses of isoquercitrin,and detect the cell viability and apoptosis(V-FITC/PI double staining).To detect the expression levels of apoptosis-associated proteins,including Bax and Bcl-2,using western blot analysis.(3)To treat the myocardial I/R-injured rats with the optimal dose of isoquercitrin,and detect the level of cell apoptosis and oxidative stress in the myocardial tissues.(4)To detect the change of mitochondrial membrane potential(MMP)in the H/R-injured H9C2 cells.To detect the expression levels of cytochrome c,caspase-9 and caspase-3 in the H/R-injured H9C2 cells.(5)To detect the expression levels of Wnt/β-cateninsignaling pathway-associated proteins,including Wnt,Frz,Dvl,β-catenin,LCF,Axin,APC,GSK-3β and CK1 using western blot analysis.(6)To overexpress Axin in H9C2 cells to investigate whether isoquercitrin exerts its anti-apoptotic role through regulating Axin/Wnt signaling Results:(1)The in vitro cellular model of myocardial I/R injury was successfully established using H/R-injured H9C2 cells.(2)Isoquercitrin administration increased the viability of H/R-injured H9C2 cells,and this effect is largely dose-dependent.(3)Isoquercitrin increased the viability of cardiomyocytes and reduced oxidative stress in myocardial I/R injured rat model.(4)Isoquercitrin decreased the apoptosis of H/R-injured H9C2 cells through restoration of MMP loss and inactivation of cytochrome c-mediated caspase pathway.(5)Isoquercitrin alleviated the I/R-induced cardiomyocyte injury through reducing the expression of Axin in Wnt/β-catenin signaling.Conclusion:Isoquercitrin treatment was found to increase the viability of I/R-injured H9C2 cells both in vivo and in vitro.Moreover,we found that isoquercitrin exerts its anti-apoptotic role through restoring the I/R-induced MMP loss and cytochrome c release.The activation of caspase-9 and caspase-3 was thus inhibited.Besides,isoquercitrin also exerts its protective role through reducing the expression of Axin in Wnt/β-catenin signaling.Collectively,our study revealed a potential cardioprotective role of isoquercitrin in alleviatingmyocardial I/R injury,which provides important experimental evidence for the treatment of this disease.