Effect Of Transcription Factor FoxO4 On Autophagy In Myocardial Hypoxia/reoxygenation Injury

Objective:To investigate the effects of transcription factor Fox O4 on autophagy in myocardial cell under hypoxia/reoxygenation condition, and explore the possible mechanism.Method:1) Take primary rat myocardial cells from postnatal 1-3 day of SD rat heart.2) Myocardial cells were respectively hypoxia for 1h, 3h, 6h, 8h, 12 h, 14 h and 24 h, also reoxygenation for 0h, 2h, 4h, 6h, 8h, 12 h and 24 h. Use western blot to observe the expression of LC3 protein and detect the LDH activity. Then choose the appropriate hypoxia and reoxygenation time point, and set up hypoxia/reoxygenation model.3) The hypoxia/reoxygenation model were hypoxia for 1h and reoxygenation for 6h. Set up five experimental groups:a) Control group: primary myocardial cells were cultured in normal culture medium.b) H/R group: primary myocardial cells were cultured for 5 days then treatment by hypoxia and reoxygenation.c) H/R +Negative virus group: primary myocardial cells were cultured for 2 days, then transfected with negative interference fragment for 3 days and treated by hypoxia and reoxygenation.d) H/R + No-load virus group: primary myocardial cells were cultured for 2 days, then transfected with no-load virus for 3 days and treated by hypoxia and reoxygenation.e) H/R+ Fox O4 si RNA virus group: primary myocardial cells were cultured for 2 days, then transfected with Fox O4 si RNA for 3 days and treated by hypoxia and reoxygenation.f) H/R+ Fox O4 overpressed virus group: primary myocardial cells were cultured for 2 days, then transfected with Fox O4 overpressed for 3 days and treated by hypoxia and reoxygenation.4) Use western blot to observe the expression of FoxO4、LC3、p62 protein and detect the LDH activity of each group. The experimental data was expressed by sx ± and T-test was performed by statistical software of IBM SPSS Statistics 20. The results in p<0.05 showed that there was significant difference.Results:1) After hypoxia for 1h、3h、6h、8h、12h、14h、24h, compared with normal control group, the expression of LC3 protein in myocardial cells were increased and at 1h it increased significantly(p<0.05); The activity of LDH also were increased, and there was significant difference compared with normal group(p<0.05).2) After hypoxia for 1h and reoxygenation for 0h、2h、4h、6h、8h、12h、24h, the expression of LC3 protein in myocardial cells were increased and at 6h it increased significantly(p<0.05); The activity of LDH also were increased, and there was significant difference compared with normal group(p<0.05).3) Primary myocardial cell was transfected by the Fox O4 and FoxO4-RNAi expression virus for 72 h, then treated by hypoxia and reoxygenation. Comparing H/R group with normal group, the expression of LDH activity was significantly increased in H/R group(p<0.05). Comparing with H/R group, the expression of LC3 prorein and LDH activity was significantly increased, also the expression of p62 prorein was significantly decreased in H/R+Fox O4 group(p<0.05); the expression of LC3 prorein and LDH activity was significantly decreased, also the p62 prorein was significantly increased in H/R+Fox O4-RNAi group(p<0.05)Conclusions:1) The level of LC3 expressed low in primary myocardial cells of normal SD rat, and expressed higher under the hypoxia/reoxygenation condition. And it was increased significantly at hypoxia for 1h and reoxygenation for 6h.2) Autophagy was excessived and myocardial cells were injuried under the hypoxia/reoxygenation condition. Up regulation of Fox O4 in myocardialcells, the excessive autophagy was further enhanced and cell damage was aggravated. Also down regulation of Fox O4 in myocardial cells, excessive autophagy and cell damage was inhibited.