The Mechanism Of P-FoxO4-Bim Signaling Contributes To Hypoxia/Reoxygenation Injury In H9c2 Cells Through Regulating Apoptosis

Part Ⅰ:Phosphorylated FoxO4(phosphorylated forkhead box subtype O4,p-FoxO4)participates in hypoxia/reoxygenation(H/R)injury of H9c2 cells.Objective To investigate whether p-FoxO4 is involved in H/R injury of H9c2 cells.Methods After 24 hours of normal culture of H9c2 cells,they were evenly inoculated into 6-well plates with a density of 4.5*10^5 cells/ml.They were randomly divided into 4 groups according to the random number table method:Control group,H9c2 cells were treated with hypoxia for 1 h reoxygenation for 1 h group(H1R1 group),hypoxia treatment for 1 h reoxygenation for 3 h group(H1R3 group),and hypoxia treatment for 1 h reoxygenation for 6h group(H1R6 group).Four groups used CCK-8 to detect the relative survival rate of cells.Real-time quantitative PCR(Real-time quantitative PCR,qPCR)method was used to detect the mRNA content of FoxO4,Bim,Bcl-2 and Bax to determine the optimal H/R time point.Hoechst staining was used to detect the degree of apoptosis in the control group and the best H/R time point group,the mitochondrial membrane potential level was detected by the JC-1 method,and the protein expression of FoxO4,p-FoxO4,Bim,Bcl-2 and Bax were detected by Western Blot.Results Compared with the Control group,the relative survival rate of H9c2 cardiomyocytes after H/R treatment was significantly reduced,of which the H1R1 group was the most significant(P<0.01).Compared with the Control group,the apoptosis rate of the H1R1 group increased(P<0.01);the mitochondrial membrane potential decreased.The mRNA levels of FoxO4,Bim and Bax in the H1R1 group were higher than those in the Control group(P<0.01),and the mRNA levels of Bcl-2 were lower than those in the Control group(P<0.01);The protein content of FoxO4,p-FoxO4,Bim and Bax was significantly higher than that of the Control group(P<0.05,P<0.01,P<0.01;P<0.05),and the protein content of Bcl-2 was significantly lower than that of the Control group(P<0.01).Conclusion p-FoxO4 promotes apoptosis and participates in hypoxia/reoxygenation injury in H9c2 cells.Part Ⅱ:p-FoxO4 participates in Hypoxia/Reoxygenation injury of H9c2 cells by regulating Bim-mediated apoptosis.Objective Silencing the FoxO4 gene and overexpressing the Bim gene further explored the molecular mechanism of p-FoxO4-Bim signal-mediated apoptosis in H9c2 cells H/R injury.Methods After cultured for 24 hours,H9c2 cells were evenly inoculated into 6-well plates and randomly divided into 3 groups according to the random number table method:hypoxia/reoxygenation group(H1R1 group),FoxO4 silencing+hypoxia/reoxygenation group(FoxO4shRNA+H1R1 group),FoxO4 silencing and Bim overexpression+hypoxia/reoxygenation group(FoxO4siRNA+Bim overexpressionRNA+H1R1 group,shFoxO4+OB+H1R1 group).The relative survival rate of cells was measured by CCK-8,the degree of apoptosis was measured by hoster staining,the mitochondrial membrane potential level was measured by JC-1 method,and the mRNA content of FoxO4,Bim,Bcl-2 and Bax was detected by qPCR,Western Blot method was used to detect the protein expression of Fox04,p-FoxO4,Bim,Bcl-2,and Bax.Results Compared with the H1R1 group,the relative survival rate of cells in the FoxO4shRNA+H1R1 group increased(P<0.01);the apoptosis rate decreased(P<0.01);the mitochondrial membrane potential improved(P<0.05);the mRNA levels of FoxO4,Bim,and Bax decreased,and the mRNA levels of Bcl-2 increased(P<0.01);FoxO4,phosphorylated FoxO4,Bim and Bax protein content decreased(P<0.01),Bcl-2 protein content increased(P<0.05).Compared with the FoxO4 shRNA+H1R1 group,the relative survival rate of the cells in the shFoxO4+OB+H1R1 group was reduced(P<0.01),the apoptosis rate was increased(P<0.01),and the mitochondrial membrane potential was significantly decreased(P<0.05),the mRNA levels of Bim and Bax were increased(P<0.01),and mRNA levels decreased(P<0.01).The protein content of Bim and Bax was significantly higher than that of the control group(P<0.01),and the protein content of Bcl-2 decreased(P<0.01).Conclusion p-FoxO4 regulates Bim-mediated apoptosis and participates in hypoxia/reoxygenation injury of H9c2 cells.