The Cardiomyocytes Protective Effects Of Quercetin Against Anoxia/reoxygenation Injury Are Mediated By ENOS

Objective:Quercetin(Que),a flavanol in the presence of food,has been confirmed improve endothelial dysfunction caused by various causes,inhibit oxidative stress and inflammation.However,there was still no clear and powerful experimental data to confirm the direct relationship betweenNOS/NO signaling pathway and quercetin pretreatment in cardiomyocytes during hypoxia-reoxygenation injury.In our study,we have established the acute myocardial anoxia/reoxygenation(A/R)injury model with H9c2 cells.We aim to investigate the effects of quercetin pretreatment onNOS/NO signaling pathway in H9c2 cardiomyocytes,cardioprotective mechanism after hypoxia-reoxygenation injury and the role of eNOS,iNOS and nNOS.Method:H9c2 cardiomyocytes was selected as subjects,and the in vitro I/R model was replicated on H9c2 cardiomyocytes by A/R treatment.MTS colorimetric assay was used to detect the survival rate of each group.The activity of lactate dehydrogenase(LDH)in the cell culture medium was detected by spectrophotometry.Western blotting was used to detect eNOS,iNOS,nNOS and anti-apoptotic protein Bcl-2.Spectrophotometry was used to detect the activity of intracellular nitric oxide(NO),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA).The level of intracellular reactive oxygen species(ROS)and the apoptosis were determined by flow cytometry.Results:1.After quercetin pretreatment,the LDH activity in the cell culture medium of the Que A/R treatment group was significantly down-regulated and the cell survival rate was significantly up-regulated,compared with the A/R group.These significant changes were dose-dependent with quercetin concentrations and The change of 20 ?M quercetin pretreatment group was the most significant;2.The detection of eNOS protein was significantly up-regulated compared with A/R group,and iNOS protein was significantly down-regulated compared with A/R group.3.The content of NO in the cells was significantly lower than that in the A/R group.4.MDA content was decreased;SOD and GSH-Px levels were increased;Intracellular ROS content was decreased significantly,Bcl-2 protein expression was significantly up-regulated and final apoptosis was decreased in 20 ?M Que A/R group Compared to A/R group;5.Compared with the Que +A/R group,the addition of the eNOS-specific inhibitor L-NAME significantly attenuated the protective effect of quercetin against cardiomyocyte hypoxia-reoxygenation injury.Conclusion:The protective effect of quercetin against A/R injury is related to the eNOS/NO signaling pathway,and at least partly eNOS protein.