The Protective Effect Of UCP2-SIRT3 Signaling Path Way On Hypxia-reoxygenation Injury Of Primary Cardiomyocytes Via Regulating Mitochondrial Oxidative Stress And Mitochondrial Dynamics

Objective In order to clarify the expression and protective effects of uncoupling protein-2(UCP2),and explore the correlation between the sirtuin-3(SIRT3)and UCP2,a hypoxia-reoxygenation(HR)injury model was established by isolated and cultured neonatal rat cardiomyocytes.To further analyze whether UCP2 regulates mitochondrial oxidative stress and mitochondrial dynamics through SIRT3 to exert myocardial protection,which provides a new research direction and lays a theoretical foundation for the research of cardioprotection against HR injury.Methods1 Establishment of HR damage model Primary cardiomyocytes were isolated and cultured for 48 hours before HR treatment.Detection the expression level of UCP2 protein and myocardial injury marker contented in cardiomyocytes.The experimental modeling time of HR time was define based on the highest protein expression of UCP2 and marked increase in myocardial injury markers.2 Evaluation of cardiomyocyte injury Test the contents of CK,LDH,cTn-T according to the instructions of the corresponding ELISA test kit.The trypan blue(TBE)staining was adopted for assessed the survival rate of cardiomyocyte.The cell counting kit-8(CCK-8)methods was used to detect cardiomyocyte viability.Apoptosis was detected by flow cytometry according to Annexin V-FITC/PI kit.3 Assessment of mitochondrial oxidative damage Detection of ROS in cardiomyocytes with the ethidium dihydrogen(DHE)fluorescent probe.Detection of Mitochondrial Membrane Potential with the JC-1 Probe.According to the ATP detection kit,the ATP content in cardiomyocytes was measured with a chemiluminescence meter.The contents of MDA,SOD and GSH in myocardial cells were detected according to the corresponding ELISA kits.4 Mitochondrial dynamics test Mitochondria were stained with the mitochondrial outer membrane receptor Tom20 and living cell mitochondrial-specific dye MitoRed,and observe and take pictures under a laser confocal microscope.Western blot(WB)method was used to detect the expression level of mitochondrial fusion protein.Results1 UCP2 could reduce HR injury in cardiomyocytes After HR injury,it was observed that the cell viability and cell survival rate were significantly reduced,and the level of myocardial injury markers in the cell culture supernatant was significantly up-regulated,which indicating that the HR model was successfully established.High UCP2 expression relieves these injuries.After UCP2 was silenced,compared with the HR group,cell damage was further aggravated.When UCP2 was silenced and UCP2 was overexpressed,it was found that the UCP2 silencing effect was reversed to a certain extent(all P<0.05).2 UCP2 reduces mitochondrial oxidative stress and mitochondrial divisionCompared with the Normoxia group,the production of ROS in HR group increasing significantly,and mitochondrial membrane potential and the production of ATP decreased markedly,the expression level of mitochondrial division-related protein increased significantly,and the expression of fusion protein expression induced markedly(all P<0.05).After constructing the model of high UCP2 expression,compared with HR group,mitochondrial oxidative damage and mitochondrial division were reduced.After UCP2was silenced,compared with the HR group,mitochondrial oxidative damage and mitochondrial division worsened.Silence UCP2 first,and then up-regulate UCP2expression,it could be found that the severity of mitochondrial oxidative damage and mitochondrial division was reversed to some extent.3 UCP2 regulates SIRT3 expression With the change of HR,the protein expression trend of SIRT3 is consistent with UCP2,and the expression is highest at H12R3 point.Compared with the Normoxia group,the content of NAD~+/NADH increased after UCP2overexpression,but significantly reduced after UCP2 silencing(P<0.05).In addition,the expression of SIRT3 Messenger RNA(mRNA)and protein was significantly increased after UCP2 overexpression(P<0.05).The expression of SIRT3 mRNA and protein was also reduced after UCP2 silencing(P<0.05),but the expression of UCP2 was no change when SIRT3 was silenced(P>0.05).Immunofluorescence detection revealed that SIRT3and UCP2 were co-localized(r=0.873,P<0.001).4 UCP2-SIRT3 reduces mitochondrial oxidative stress and mitochondrial divisionThrough further research,we could observed that compared with the HR group,the degree of cardiomyocyte injury and mitochondrial oxidative stress and division were further exacerbated when SIRT3 was silenced.However,by down-regulating SIRT3 and up-regulating UCP2,it could be revealed that the degree of damage to the former was not significantly reversed(P>0.05),which further indicating that the myocardial protection of UCP2 depends on the presence of SIRT3.Conclusion UCP2 has myocardial protective effect in reducing HR injury.UCP2 plays an important role in reducing oxidative damage to cardiomyocytes and mitochondrial division,and the function of stabilizing mitochondrial homeostasis.UCP2 may play a protective role by acting on SIRT3,that is,the UCP2-SIRT3 pathway exerts protective effects on HR-induced myocardial injury by regulating cellular oxidative stress and mitochondrial dynamics(division or fusion).