Abstract: Objective To investigate the effects of phosphorylated forkhead box subtype O4 (p‑FoxO4) in H9c2 cells with hypoxia/reoxygenation (H/R) injury. Methods H9c2 cells were cultured in 6‑well plates for 24 h at a density of 4.5×105 cells/ml. Each group was repeated more than or equal to three times. They were divided into four groups according to the random number table method: a control group, a hypoxia 1 h/reoxygenation 1 h (H1R1) group, a hypoxia 1 h/reoxygenation 3 h (H1R3) group and a hypoxia 1 h/reoxygenation 6 h (H1R6) group. The relative survival rate of the cells was detected by the cell proliferation‑toxicity detection method. The mRNA contents of forkhead box subtype O4 (FoxO4), Bcl‑2 interacting mediator of cell death (Bim), B‑cell lymphoma‑2 (Bcl‑2) and Bcl‑2‑Associated X (Bax) were detected by real‑time quantitative PCR (qPCR) to determine the optimal H/R time point. The apoptosis in the control group and the H1R1 group was detected by Hoechst staining. The levels of FoxO4, p‑FoxO4, Bim, Bcl‑2 and Bax were measured by Western blot. According to the random number table method, C57BL/6 mice aged 6 to 8 weeks were divided into five groups (n=3): a sham operation (sham) group, an ischemia 30 min and reperfusion 3 h (R3) group, an ischemia 30 min and reperfusion 6 h (R6) group, an ischemia 30 min and reperfusion 12 h (R12) group, and an ischemia 30 min and reperfusion 24 h (R24) group. The mRNA contents of FoxO4, Bim, Bcl‑2 and Bax in the anterior wall of the left ventricle in mice with ischemia after ligation of the left anterior descending branch were detected by qPCR. Results Compared with the control group, the relative survival rate of cells in the H1R1, H1R3 and H1R6 groups decreased, where the HIR1 group was the lowest (P<0.05). Compared with the H1R1 group, the relative survival rates of the cells in the H1R3 and H1R6 groups increased (P<0.05). Compared with the H1R3 group, the relative survival rate of the cells in the H1R6 group increased (P<0.05). Compared with the control group, the levels of FoxO4 mRNA increased in the H1R1, H1R3, and H1R6 groups (P<0.05), the levels of Bim mRNA increased in the H1R1 group (P<0.05), the levels of Bcl‑2 mRNA decreased in the H1R1 group (P<0.05), and the levels of Bax mRNA increased in the H1R1 group (P<0.05), and the levels of Bax mRNA decreased in the H1R3 and H1R6 groups (P<0.05). Compared with the H1R1 group, the levels of Bim and Bax mRNA decreased in the H1R3 and H1R6 groups (P<0.05). Compared with the sham group, FoxO4 mRNA content increased in the R12 and R24 groups (P<0.05), Bim mRNA decreased in the R6 group (P<0.05), and Bim mRNA content increased in the R24 group (P<0.05), Bcl‑2 mRNA content decreased in the R3, R12, and R24 groups (P<0.05); Bax mRNA content increased in the R6 and R24 groups (P<0.05). Compared with the R3 group, Fox04 mRNA content increased in the R12 and R24 groups (P<0.05), Bim mRNA content increased in the R12 and R24 groups (P<0.05), and Bax mRNA content increased in the R6, R12 and R24 groups (P<0.05). Compared with the R6 group, FoxO4 and Bim mRNA content increased in the R12 and R24 group (P<0.05); Compared with the R12 group, FoxO4, Bim and Bax mRNA content increased in the R24 group (P<0.05). Compared with the control group, there were more nuclear pyknosis and bright apoptotic cells in the H1R1 group, and the difference in apoptotic rate was statistically significant (P<0.05). Compared with the control group, the contents of FoxO4, p‑FoxO4, Bim, and Bax protein in the H1R1 group increased, while the content of Bcl‑2 protein decreased (P<0.05). Conclusions p‑FoxO4 may be involved in H/R injury of H9c2 cells through regulating the intrinsic pathway of apoptosis.
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