FOXO4 Promotes Apoptosis Via Chop In H9C2 Cell Hypoxia/Reoxygenation

Objective:To study the relationship between FOXO4 and Chop dependent apoptotic pathway of H9C2 cells in hypoxia/reoxygenation(H/R).Methods:The H9C2 cells were cultured in vitro and the H/R model of H9C2 cells was established.The study included three parts.In the first part,the cells were randomly divided into four groups: the normal group,the only H/R group,the H/R plus negative virus group and the H/R plus FOXO4-siRNA virus group.In the second part,the cells were randomly divided into two groups: H/R plus negative interference fragment group,the H/R plus Chop siRNA group.In the third part,the cells were randomly divided into four groups: the normal group,the only H/R group,the H/R plus negative virus and control plasmid group and the H/R plus FOXO4-siRNA virus and Chop overexpressing plasmid group.TUNEL was used to measure the levels of apoptotic.The protein levels of FOXO4、Chop、Cleaved-caspase3 were measured by Western Blot.CHIP was used to measure binding site between FOXO4 and Chop.Results:(1)Western Blot showed that the protein levels of FOXO4,Chop and Cleaved-caspase3 were significantly higher in hypoxia/reoxygenation,and TUNEL showed that the levels of apoptotic was significantly higher in hypoxia/ reoxygenation.(2)Compared with the H/R plus negative virus group,pretreatment with FOXO4-siRNA reduced the protein expressions of FOXO4,Chop,Cleaved-caspase3(P<0.05);TUNEL showed that the levels of apoptotic was significantly lower(P<0.05).Pretreatment with Chop-siRNA reduced the protein expressions of Chop,Cleaved-caspase3(P<0.05)(3)The H/R plus FOXO4-siRNA virus and Chop overexpressing plasmid group had lower protein levels of FOXO4(P<0.05)and higher protein levels of Chop,Cleaved-caspase3(P<0.05)compared with the H/R plus negative virus and control plasmid group(P<0.05).(4)CHIP showed that compared with the normal group,pretreatment with H/R significantly activate FOXO4 bind to the promoter region of Chop,the difference has statistical significance(P<0.05).Conclusion:(1)The expressions of FOXO4、Chop and Cleaved-caspase3 of H9C2 cells were Significantly increased in hypoxia/reoxygenation,and the levels of apoptotic was also significantly increased.Inhibition of FOXO4 can downregulate the expressions of FOXO4、Chop and Cleaved-caspase3 in hypoxia/reoxygenation.(2)Inhibition of Chop can downregulate the expressions of Chop and Cleaved-caspase3 in hypoxia/reoxygenation.Inhibition of FOXO4 and overexpression of Chop can downregulate the expressions of FOXO4 and upregulate the expressions of Chop and Cleaved-caspase3 in hypoxia/reoxygenation.(3)CHIP showed that FOXO4 bind to the promoter region of Chop,as FOXO4 direct target with Chop.FOXO4-Chop axis may be a therapeutic target for protecting myocardial ischemia-reperfusion injury.