| [Objective] In this study,primary myocardial cells are culturing and the pathological process of myocardial infarction was simulated by constructing mouse myocardial infarct model,the regulation mechanism of silencing information regulator 1(Sirtuin1,Sirt1)in myocardial apoptosis induced by hypoxia in acute myocardial infarction was investigated.[Methods](1)Acute myocardial infarction mice were constructed by ligating the left anterior descending coronary artery of mice,and the infarction and peripheral tissue were collected at different time after myocardial infarction(1,3 and 7 days)(the myocardial tissue of the sham operation group was taken as the control),and the expression of Sirt1 was detected by RT-PCR and Western blot.(2)Sirt1 overexpressed lentivirus was constructed,and Sirt1 overexpressed lentivirus was injected into the myocardial infarction zone of the mouse myocardial infarction model.Meanwhile,the negative lentiviral dot injection group was used as the control group.7 days after the surgery,the mouse heart function was detected and the myocardial infarction area was detected by TTC method.(3)Primary mouse myocardial cells were cultured in vitro,Western blot was used to detect the expression levels of Sirt1 and Cleaved-caspase3 protein in cells after different hypoxia time,and TUNEL method was used to detect the effect of Sirt1 expression on apoptosis of myocardial cells.(4)Western blot was used to detect the expression levels of PHD3 and hif-1 protein in myocardial cells at different time of hypoxia,and immunofluorescence was used to detect the cell sites and expression changes of PHD3.(5)To investigate the effect of Sirt1 on the PHD3/ hif-1 signaling pathway,primary myocardial cells of mice were cultured in vitro,and Sirt1 overexpressed lentivirus and small interfering RNA were used to stimulate myocardial cells under the condition of normoxia and hypoxia,respectively,and the expression changes of Sirt1 PHD3 and hif-1 were detected by Western blot.(6)A mouse model of acute myocardial infarction was constructed.Sirt1 overexpressed lentivirus was injected into the myocardial infarction site in mice,and the negative lentivirus site injection group was used as the control group.The expression changes of Sirt1 and PHD3 were detected by immunohistochemistry 7 days after surgery,and the apoptosis of myocardial cells was detected by TUNEL assay.[results](1)m RNA and protein expression levels of Sirt1 in myocardial infarction and peripheral tissues were significantly decreased between 1 and 7 days after infarction(P<0.05).Meanwhile,compared with the negative control group,the Sirt1 overexpression lentiviral injection group significantly decreased the infarct area(15.68±3.28 vs.29.50±5.56,P<0.05).(2)Compared with the control group,Sirt1 protein expression level decreased and Cleaved-caspase3 protein expression level increased after different time of hypoxia.The results were statistically significant(P<0.05).Overexpression of Sirt1 reduced hypoxiainduced myocardial Apoptosis.(3)Compared with the control group,the expression level of PHD3 protein increased and the expression level of HIF-1α protein decreased after different time of hypoxia.The results were statistically significant(P<0.05).(4)Under normoxic and hypoxic conditions,myocardial cells transfected with Sirt1 overexpressing lentivirus can activate the expression of Sirt1,and at the same time activate the expression of PHD3,and the expression of hif1-is affected and decreased.Myocardial cells transfected with small interfering RNA inhibited the expression of Sirt1 and PHD3,and increased expression of HIF-1α showed statistically significant differences(P<0.05).(5)In the myocardial infarction tissue,the expression levels of Sirt1 and PHD3 in the Sirt1 overexpression lentivirus group were increased compared with the negative lentivirus control group;Compared with the negative lentivirus control group,the overexpressed Sirt1 lentivirus group reduced the apoptosis of myocardial cells(P<0.05).[Conclusion] Sirt1 can reduce the apoptosis of myocardial cells after hypoxia and the area of myocardial infarction.Sirt1 participates in the pathological process of myocardial infarction through the influence of PHD3/HIF-1α signaling pathway and reduces the apoptosis of cardiomyocytes. |
