Myocardial Protective Effects And Molecular Mechanisms Of Natural Flavone Acacetin

Structural remodeling and functional disturbances of the heart,caused by different types of heart diseases,may finally lead to cardiac dysfunction and heart failure,which are the major causes of high cardiovascular mortality around the world.Developing novel therapeutic approaches of myocardial disorders are the important strategy to reduce the cardiovascular mortality.The aim of the present PhD project is to investigate the molecular signal pathway of the natural flavone acacetin in cardioprotection against ischemia/reperfusion injury and to explore whether acacetin can treat cardiomyopathy induced by the anti-cancer drug doxorubicin using multiple experimental approaches of biochemistry and molecular biology.In first part of the study,the potential molecular mechanism of acacetin in cardioprotection against ischemia/reperfusion injury using a cell model of hypoxia/reoxygenation injury in primary cultured neonatal rat cardiomyocytes and H9C2 cardiomyoblasts.It was found that acacetin(0.3-3 μM)significantly decreased the apoptosis and ROS production induced by hypoxia/reoxygenation injury in cardiomyocytes and H9C2 cardiomyoblasts via reducing the pro-apoptotic proteins Bax and cleaved-caspase-3 and increasing the anti-apoptotic protein Bcl-2.In addition,acacetin not only suppressed the release of pro-inflammatory cytokines TLR-4 and IL-6 induced by hypoxia/reoxygenation injury,but also increased the secretion of anti-inflammatory cytokine IL-10.Moreover,acacetin increased Nrf2 and HO-1 in a concentration-dependent manner,and rescued SOD1 and SOD2 reduction induced by hypoxia/reoxygenation insult.These beneficial effects of acacetin disappeared in cells with silenced Nrf2,suggesting that Nrf2 activation participates in the cardioprotective effect of acacetin against hypoxia/reoxygenation insult.However,acacetin-induced Nrf2 activation was not observed in cells with silenced AMPK.Our results demonstrate that AMPK-mediated Nrf2 activation is involved in the cardiomyocytes protection of acacetin against hypoxia/reoxygenation injury by activating a series of intracellular signals involved in anti-oxidation,anti-inflammation,and anti-apoptosis.In second part of the study,a cell model of doxorubicin-induced cardiomyocytes injury was produced in H9C2 cardiomyoblasts and an animal model of doxorubicin-induced cardiomyopathy was established in mice.Doxorubicin at 1 μM significantly decreased cell viability by increasing ROS generation and apoptosis via reducing intracellular antiapoptotic proteins.Acacetin at 0.3-3μM antagonized doxorubicin-induced viability reduction,ROS generation and apoptosis via stimulating Sirtl/AMPK,then activating antioxidation pathway of Nrf2/HO-1 and SOD1/SOD2.Acacetin prodrug(15 mg/kg,subcutaneously,b.i.d.)significantly increased survival rate,improved the reduction of ejection fraction and fractional shortening,and the increase of ventricular end-diastolic dimension in doxorubicin-induced cardiomyopathy in mice.Histological and biochemical analysis revealed that acacetin descreased myocardial fibrosis and activated myocardial Sirtl/AMPK and increased anti-oxidants.Collectively,my PhD study demonstrates for the first time that that 1)AMPK-mediated Nrf2 activation is involved in cardiomyocytes protection of acacetin against hypoxia/reoxygenation injury;2)activation of Nrf2 by acacetinis medated by Sirtl/AMPK pathway confers cardioprotection against doxorubicin-induced cardiomyopathy.Therefore,acacetin is a new member of Sirtl/AMPK activators,which may be a promising drug candidate for treating cardiomyopathy induced by ischemia/reperfusion or anti-cancer drugs.