The Effect And Mechanism Of Sestrin2 On Rat Cardiomyocytes Hypoxia/Reoxygenation Injury

BackgroundThe mechanism of myocardial reperfusion injury has not been fully elucidated,which is one of the clinical problems need to be solved urgently.Previous studies have shown that it is closely related to oxidative stress,autophagy,energy metabolism disorder,neutrophil inf iltration and so on.Oxidative stress plays an important role in ischemia/reperfusion injury.As a newly discovered protein,Sestrin2 plays an important role in reducing the accumulation of reactive oxygen species(ROS)and maintaining energy balance.Recently,Sestrin2 protein plays a protective role in cerebral ischemia/reperfusion by inducing autophagy and a ctivating AMPK signal pathway,which may be an important potential protective factor in vivo.Nrf2 related signaling pathway is one of the most importantend ogenous antioxidant stress pathways at present.It plays an antioxidantrole by regulating antioxidant response elements and activating down stream antioxidant factors,such as GST,HO-1,NQO-1,SOD and so on.A number of studies have shown that the activation of Nrf2 and its downstream antioxidant genes can protect against myocardialisch emia/reperfusion injury.However,the role of Sestrin2 protein in cardiomyocyte ischemia/reperfusion and its relationship with Nrf2-ARE signal pathway have not been reported.Because the pathophysiological process of hypoxia/reoxygenation of cardiomyocytes is similar to isch emia/reperfusion of human cardiomyocytes,we intend to use hypoxia/reperfusion injury of rats' H9C2 cardiomyocytes to simulate the process of myocardial ischemia/reperfusion.ObjectTo investigate the effect and mechanism of Sestrin2 on rat cardiom yocyte hypoxia/reoxygenation injury.MethodsH9C2 cardiomyocytes were cultured and constructed hypoxia/reoxy genation injury(H/R)models in vitro.Cell survival rate was measured by cell counting kit-8(CCK8);Annexin V/7-AAD staining with flow cytometry was used to detect apoptosis.Then H9C2 cardiomyocytes were divided into normal control group,hypoxia 4h group,hypoxia 4h group,hypoxia/reoxygenation 4h+ si-NC group and hypoxia/reoxygenation 4h+siRNA group.Lactate dehydrogenase(LDH)as a marker for myocardial cell injury was measured by Lactate dehydrogenase assay kit.A recombinant siRNA(siRNA)for Sestrin2 silencing was constructed and tr ansfected into H9C2 cardiomyocytes with liposomes.The expression of Sestrin2 protein was detected by Western blot.Flow cytometry was used to investigate the apoptosis.Using Western blot to detect the expres sion of Sestrin2,Nrf2 and HO-1.Results1 Compared with the normal control group,the CCK8 results showed that the cell survival rate of H4/R4 group was decreased,and the flow cytometry results showed that the apoptosis rate was significantly increased(P< 0.05),LDH level was increased(P<0.01).2 Compared with the normal control group,all of the expression of Sestrin2 in hypoxia 4 h group and H4/R4 group were increased,and the expression in H4/R4 group was higher than hypoxia 4h group(P< 0 01).3 After down-regulating the expression of Sestrin2 specifically and effectively by siRNA,the apoptosis was significantly increased compared with the corresponding control group(P< 0.05).At the same time,the expression of Nrf2 protein and HO-1 protein decreased.Conclusion1 Hypoxia and reoxygenation can reduce cells injury and much more serious after reoxygenation.2 The protect effect of Sestrin2 in myocardial ischemia reperfusion injury may be relevant to Nrf2/HO-1 signaling pathway.