UCP2 Overexpression Regulates Mitochondrial Autophagy To Alleviate Hypoxia-reoxygenation Injury Of H9C2 Cardiomyocytes

Objective H9C2 cardiomyocytes were used to establish hypoxia reoxygenation(HR)injury model.To observe the effect of hypoxia reoxygenation injury on the expression of uncoupling protein-2(UCP2)in cardiomyocytes,clarify the myocardial protective effect of UCP2,and further explore the relationship between the myocardial protective effect of UCP2 and mitochondrial autophagy,so as to provide a new research direction for the research on the myocardial protective effect of HR induced injury.Methods1 Establishment of hypoxia-reoxygenation injury model H9C2 cardiomyocytes were subcultured for 24-36 hours,and hypoxia and reoxygenation were performed when the cell fusion reached about 80%.The expression of UCP2 protein at different time points and the changes of myocardial injury markers were detected.In this experiment,the hypoxia-reoxygenation time point when the UCP2 protein level is the highest,the myocardial cell viability is the lowest,and the myocardial injury markers are significantly increased are selected as the HR injury model modeling time.2 Evaluation of cardiomyocyte injury Cardiomyocyte damage markers c Tn-T,LDH,CK-MB were detected by ELISA kit and according to the instructions.The activity of myocardial cells was detected by CCK8 assay.Cell apoptosis was detected by flow cytometry.3 To evaluate mitochondrial oxidative damage Determination of Reactive Oxygen Species(ROS)in Cardiomyocytes by Fluorescent Probe of Dihydroethidium(DHE).4 Cell and mitochondrial autophagy detection Western blot and immunofluorescence detection of cell autophagy protein and mitophagy protein expression,transmission electron microscope observation of mitophagy.Results1 UCP2 attenuates hypoxia-reoxygenation injury of cardiomyocytes The detection of H9C2 cardiomyocytes after hypoxia-reoxygenation injury showed that the levels of myocardial injury markers increased significantly,and the cell survival rate and cell viability decreased significantly,indicating that the hypoxia-reoxygenation model was successfully established.Cell damage markers,cell survival rate,and cell viability were further reduced after UCP2 was silenced.After UCP2 overexpression,the level of cell damage markers decreased,while cell survival rate and cell viability increased,indicating that UCP2 reduced myocardial cell hypoxia and reoxygenation damage.2 UCP2 regulates mitophagy and reduces HR damage of cardiomyocytes After cell hypoxia and reoxygenation,the expression of mitochondrial autophagy protein was increased compared with the normal group(Normoxia).When UCP2 was silenced,the expression of mitochondrial autophagy protein was further increased compared with the hypoxia-reoxygenation group.After UCP2 overexpression,the mitochondrial autophagy protein was lower than the hypoxia-reoxygenation group and silence group(all P<0.05).These results suggest that the decreased level of mitochondrial autophagy may be involved in the cardiac protective effect of UCP2 in cardiac I/R injury.Conclusion1.Silencing of UCP2 can aggravate the HR damage of cardiomyocytes,on the contrary,overexpression has a certain protective effect;2.Mitophagy participates in the maintenance of cardiomyocyte homeostasis after HR injury;3.UCP2 overexpression exerts an anti-HR injury effect on cardiomyocytes by reducing the level of Mitophagy.