异氟醚对高糖诱导的H9C2心肌细胞损伤及PI3K/Akt/mTOR信号通路的影响

Effect of Isoflurane on High Glucose-induced Cardiomyocyte Injury in H9C2 via PI3K/Akt/mTOR Signaling Pathway

目的探讨异氟醚对高糖诱导的H9C2心肌细胞损伤及磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路的影响。方法选取对数生长期的H9C2心肌细胞,分组为:①对照组,未处理的H9C2心肌细胞;②高糖组(35 mmol/L葡萄糖);③异氟醚低浓度组(在高糖组的基础上加入1%异氟醚);④异氟醚高浓度组(在高糖组的基础上加入2%异氟醚);⑤异氟醚高浓度+LY294002组(在高糖组的基础上加入2%异氟醚+50μmol/L LY294002)。CCK-8法检测各组细胞增殖;透射电子显微镜观察各组细胞自噬情况;流式细胞术检测各组细胞凋亡;Western印迹检测各组细胞中PI3K/Akt/mTOR通路及自噬(Beclin1)、凋亡(Bax)相关蛋白表达。结果与对照组相比,高糖组细胞中明显可见形成的自噬小体,增殖率、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR降低,细胞凋亡率、Bax、Beclin1蛋白表达显著增加(P<0.05)。与高糖组相比,经不同浓度的异氟醚处理后,细胞增殖率、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR显著增加,自噬小体减少,细胞凋亡率、Bax、Beclin1蛋白表达显著下降(P<0.05);与异氟醚高浓度组相比,异氟醚高浓度+LY294002组细胞增殖率、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR降低,自噬小体增多,细胞凋亡率、Bax、Beclin1蛋白表达显著增加(P<0.05)。结论异氟醚可以抑制高糖诱导的H9C2心肌细胞自噬、凋亡,提高细胞存活率,减少细胞损伤,可能与PI3K/Akt/mTOR通路的激活有关。

Objective To investigate the influences of isoflurane on high glucose-induced cardiomyocyte injury in H9C2 cells and its mechanism through phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signaling pathway.Methods H9C2 cardiomyocytes in the logarithmic growth phase were selected and grouped into 5 groups:control group(untreated H9C2 cardiomyocytes),high glucose group(35 mmol/L glucose),isoflurane low-dose group(35 mmol/L glucose+1%isoflurane),isoflurane high-dose group(35 mmol/L glucose+2%isoflurane),isoflurane+LY294002 group(35 mmol/L glucose+2%isoflurane+50μmol/L LY294002).The proliferation of cells in each group was detected by CCK-8 method.The autophagy of cells in each group was observed by transmission electron microscope.The apoptosis of cells in each group was detected by flow cytometry.The expression levels of Beclin1,Bax,and PI3K/Akt/mTOR pathway related proteins in the cells of each group were measured by Western blotting.Results The autophagosomes formed in the cells of the high glucose group could be observed obviously.The proliferation rate,and the levels of p-PI3K/PI3K,p-Akt/Akt,and pmTOR/mTOR in high glucose group were decreased,while the apoptosis rate,and the expression levels of Bax and Beclin1 proteins were increased significantly when compared with those in the control group(P<0.05).After treated with different concentrations of isoflurane,the cell proliferation rate,and the levels of p-PI3K/PI3K,p-Akt/Akt and p-mTOR/mTOR were increased significantly,while the numbers of autophagosomes,the apoptosis rate,and expression levels of Bax and Beclin1 proteins were decreased significantly when compared with those in the high glucose group(P<0.05).The cell proliferation rate,levels of p-PI3K/PI3K,p-Akt/Akt and p-mTOR/mTOR decreased in the isoflurane+LY294002 group when compared with those in the isoflurane high-dose group,while the number of autophagosomes,the apoptosis rate,and the expression levels of Bax and Beclin1 proteins were increased significantly(P<0.05).Conclusion Isoflurane can inhibit the high-glucose induced autophagy and apoptosis in the H9C2 cardiomyocytes,which improves cell survival rate and reduces cell injury.The underlying mechanism may be related to the activation of PI3K/Akt/mTOR pathway.