| Object:This study was to investigate the effect of overexpression of uncoupling protein2 both on isoprenaline-induced H9C2 cardiomyocyte hypertrophy and related mechanisms.Method:1.Using isoprenaline to induce hypertrophy of H9C2 cardiomyocytes,we used isoproterenol to treat serum-starved H9C2 cells for 48 hours and divided them into normal group and isoproterenol concentration of 5 ?mol/L,25 ?mol/L,50 ?mol./L group.Then reactive oxygen species were detected by flow cytometry,as well as the survival rate of each group in cells detected by CCK8 assay.Real-time reverse transcription polymerase chain reaction was used to detect the expression of cardiomyocyte hypertrophy markers such as Acta1 mRNA,ANP mRNA,Myh7 mRNA,and UCP2 mRNA in each group.2.Overexpression of UCP2 adeno-associated virus successfully transfected H9C2 cardiomyocytes.Then we divided them into negative control group,rAAV9 group and rAAV9-UCP2 group respectively.After adeno-associated virus was transfected into cardiomyocytes for 72 hours,not noly was transfection efficiency examined under inverted fluorescence microscope,but UCP2 protein expression was also detected by Western blot.3.In order to observe the effect of overexpression of UCP2 on cardiomyocyte hypertrophy and related mechanisms,we divided the cells into 4 groups: normal group,over-expressing UCP2 group,ISO group and over-expressing UCP2 adding ISO group.The reactive oxygen species was detected by flow cytometry and the expression of Acta1 mRNA,ANP mRNA,and Myh7 mRNA was detected by RT-PCR.At the same time,the expression of cytoskeletal protein ?-actinin was detected by cellular immunofluorescence,while the expression of protein PI3 K,AKT,p-AKT,Gsk3?,p-Gsk3?,Grp78 was detected by Western blot.Result:1.We investigated the effect of ISO on ROS generation and cell survival rate in H9C2 cardiomyocytes.The ROS level including ISO 5?mol/L group,ISO 25?mol/L group,ISO 50?mol/Lgroup and normal group were 1848.7±23.46,2311.3±62.15,2493 ± 8.5,1508 ± 23.46 respectively(comparison between groups,P<0.01).This shows that as the concentration of ISO increases,so does the level of ROS.In addition,as the concentration of ISO increased,the cell survival rate gradually decreased.2.Isoprenaline successfully induced hypertrophy of H9C2 cardiomyocytes.Compared with the control group,the expression of cardiomyocyte hypertrophy markers such as Acta1 mRNA,ANP mRNA,and Myh7 mRNA both in the ISO 25?mol/L and ISO 50 ?mol/L groups increased(P<0.01).3.In this experiment,adeno-associated virus was used to successfully transfect H9C2 cardiomyocytes.observing the transfection efficiency under a fluorescence microscope,it reached 75% or more.The results of WB test showed that UCP2 protein expression was increased in UCP2 transfection group compared with the empty group(P<0.01).4.We investigated the effect of overexpression of UCP2 on myocardial ROS levels and cardiomyocyte hypertrophy.The experimental results show that overexpression of UCP2 can reduce the level of reactive oxygen species produced by isoproterenol intervention and the expression levels of cardiomyocyte hypertrophy markers such as Acta1 mRNA,ANP mRNA,and Myh7 m RNA(2.45±0.18,1.72±0.068,1.6).±0.1 vs 3.82±0.07,3.31±0.34,2.63±0.35,P<0.01);At the same time,with overexpression of UCP2,the expression of ?-actinin was decreased,which indirectly responded that the area of myocardial hypertrophy was decreased(P<0.01).5.We investigated the mechanism of UCP2 in inhibiting cardiomyocytes hypertrophy and its effect on endoplasmic reticulum stress.The results showed that overexpression of UCP2 reduced the levels of PI3 K,P-Akt,P-GSK3?,and Grp78 protein in isoproterenol-induced cardiomyocytes hypertrophy.Conclusion:1.Not only overexpression of uncoupling protein 2 reduces the reactive oxygen species in H9C2 cardiomyocytes,but also the levels of cardiomyocytes hypertrophy markers.2.Uncoupling protein 2 may inhibit cardiomyocytes hypertrophy by inhibiting Akt/Gsk-3? signaling pathway.3.Uncoupling protein 2 may reduce endoplasmic reticulum stress. |
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