| Background and Aim:It is often accompanied by cardiac dysfunction in the development of various cardiovascular diseases such as myocardial ischemia/reperfusion injury,cardiomyopathy,myocardial hypertrophy,and heart failure.Cardiomyocyte injury is an important pathological basis.Inhibition of oxidative stress,calcium overload,cardiomyocyte apoptosis and so on,then improving cardiac dysfunction,is the important research direction of cardiac protection.Homocysteine(Hcy),as an intermediate metabolite of the methionine cycle in the body,is an independent risk factor for cardiovascular diseases.Persistent high concentration of serum Hcy can induce oxidative stress and cardiomyocytes apoptosis,promote myocardial fibrosis,accelerate cardiac remodeling,cause to myocardial injury,and eventually lead to cardiac dysfunction.Puerarin(Pue)is an isoflavone derivative isolated from the traditional Chinese medicine pueraria lobata root.It can reduce myocardial injury induced by multiple risk factors and improve cardiac function through anti-oxidation,anti-myocardial apoptosis and anti-myocardial fibrosis.It is also benefical in protecting ischemic myocardium,reducing myocardial ischemia reperfusion injury and the area of acute myocardial infarction.However,there is no study to explore the protective effect of Pue on Hcy-induced cardiomyocytes injury and the possible molecular mechanism of this effect.This study was to explore the protective effect of Pue on Hcyinduced H9c2 cell injury and its possible mechanism,and provide new ideas for the application of Pue in the cardiac protection.Methods:Rat cardiomyocyte H9c2 cells were cultured in vitro and treated with different concentrations of Hcy(0～8 mM)for 24 h.The morphological changes of H9c2 cells were observed under light microscope;The cell viability and cytotoxicity were detected by CCK-8 kit and LDH kit respectively;The rate of apoptosis was measured by flow cytomety with Annexin-V FITC/PI double staining method;The level of ROS in cells was tested through the microplate method.Pue was pre-treated for the following intervention experiments,and the same method was used to measure the changes in the above detection.In addition,TBA method was used to detect intracellular malondialdehyde(MDA)level;WST method was used to detect intracellular total superoxide dismutase(SOD)level;Seahorse bioanalyzer was used to detect mitochondrial respiratory function;The mRNA expression levels of Nrf2 and HO-1 were measured by real-time quantitative PCR;The protein expression levels of Akt,pAkt,Nrf2,HO-1,Bax,Bcl-2 and cleaved caspase-3 were detected by Western blot.Finally,using HO-1 specific blocker(ZnPP)and PI3K specific blocker(LY294002)to block HO-1 and inhibit the activation of PI3K respectively.The possible mechanism of Pue inhibiting Hcy induced H9c2 cardiomyocyte injury was explored.Results:(1)Hcy could decrease the viability of H9c2 cells,increase the release of LDH,cause oxidative damage and induce cell apoptosis in a concentration-dependent manner;(2)Pretreatment with Pue could increase the viability of H9c2 cells,reduce LDH leakage and the rate of apoptosis,increase SOD level,decrease MDA level,reduce oxidative damage and improve mitochondrial respiratory function.At the same time,Pue could reduce the ratio of Bax/Bcl-2 and the expression of cleaved caspase-3,and promote the expression of Nrf2 and HO-1 mRNA and protein;(3)Hcy could reduce the expression of p-Akt,while pretreatment with Pue could increase the expression of p-Akt.PI3K inhibitor(LY294002)could block the effects of Pue to increase the expression of Nrf2 and HO-1 protein and increase cell viability;HO-1 inhibitor(Znpp)could reverse the effects of Pue in reducing the ratio of Bax/Bcl-2,cleaved caspase-3 expression and increasing cell viability.Conclusion:In this study,we successfully established a model of H9c2 cardiomyocyte injury induced by Hcy.Pue pretreatment could inhibit H9c2 cardiomyocyte injury induced by Hcy and improve myocardial mitochondrial respiratory function.Pue could protect myocardium by reducing oxidative stress and inhibiting apoptosis,which may be related to the activation of PI3K/Akt-Nrf2/HO-1 signaling pathway. |
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