Effects of autophagy-related protein 5 on mitophagy in oxygen-glucose deprivation/reoxygenation injured cardiomyocytes
Objective To observe the effect of autophagy-related protein 5(ATG5)on mitophagy in oxygen-glucose deprivation/reoxygenation(OGD/R)-induced H9c2 cardiomyocytes.Methods OGD/R was used to simulate my-ocardial ischemia-reperfusion injury in H9c2 cardiomyocytes.H9c2 cells were treated as follows:no treatment(con-trol group),OGD/R model(OGD/R group),OGD/R+transfection of empty vector(OGD/R-Vehicle group),OGD/R+transfection of ATG5 overexpression plasmid(OGD/R-ATG5 group),OGD/R+meaningless sequence interference RNA(OGD/R-siRNA-negative group)and OGD/R+silencing ATG5 gene sequence(OGD/R-siRNA-ATG5 group).Cell counting kit 8(CCK8)was used to detect the proliferation of H9c2 cells.Mitochondrial survival was observed under microscope.Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and west-ern-blot were used to detect the mRNA and protein expressions of ATG5,FUN14 domain-containing protein 1(FUNDC1),dynamin-related protein 1(DRP1),and optic atrophy associated protein 1(OPA1).Western-blot was used to detect the expressions of microtubule-associated protein 1A/1B-light chain 3 Ⅱ(LC3 Ⅱ)and nuclear porin glycoprotein P62(P62).Results Compared with the control group,the proliferation rate of H9c2 cells in OGD/R group,OGD/R-Vehicle group,OGD/R-ATG5 group,OGD/R-siRNA negative group and OGD/R-siRNA-ATG5 group was decreased(F=106.35,P＜0.05).Mitochondrial activity was decreased in OGD/R,OGD/R-vehicle,OGD/R-siRNA negative and OGD/R-siRNA-ATG5 groups,while increased in OGD/R-ATG5 group(F=50.74,P＜0.05).The expression levels of ATG5,FUNDC1 and DRP1 mRNA and protein in OGD/R group,OGD/R-ve-hicle group,OGD/R-ATG5 group and OGD/R-siRNA negative group were increased,while the mRNA and protein expressions of ATG5,FUNDC1 and DRP1 were decreased in OGD/R-siRNA-ATG5 group(all P＜0.05).The ex-pression levels of OPA1 mRNA and protein in all groups were lower than those in the control group(F=9.924,24.334,P＜0.05).The expression level of LC3 Ⅱ protein in OGD/R group,OGD/R-Vehicle group,OGD/R-ATG5 group and OGD/R-siRNA negative group was increased,and the expression level of P62 protein was de-creased(F=18.07,17.50,P＜0.05).In the OGD/R-siRNA-ATG5 group,the expression level of LC3 Ⅱ protein was decreased,and the expression level of P62 protein was increased.Compared with group OGD/R,the cell prolif-eration rate,mitochondrial activity,mRNA and protein expressions of ATG5,FUNDC1,DRP1 and OPA1 were significantly increased,the protein expression of LC3 Ⅱ was increased,and the protein expression of P62 was de-creased in group OGD/R-ATG5.In the OGD/R-siRNA-ATG5 group,the cell proliferation rate,mitochondrial ac-tivity,mRNA and protein expressions of ATG5,FUNDC1,and OPA1,DRP1 protein expression,LC3 Ⅱ protein expression,and P62 protein expression were decreased(all P＜0.05).There was no significant difference in all in-dexes between OGD/R-vehicle group and OGD/R group,OGD/R-siRNA negative group and OGD/R group(P＞0.05).Conclusion H9c2 cells show mitochondrial fission and mitophagy after OGD/R injury,and ATG5 can en-hance mitophagy to alleviate OGD/R injury in cardiomyocytes.
ischemia reperfusion injurymitophagymitochondrial fissionautophagy related protein 5car-diomyocytes H9c2
万方数据
维普
