细胞色素P450表氧化酶通过PI3K/AKT及ERK/MAPK途径抗内皮细胞凋亡的机制

目的:研究细胞色素P450表氧化酶对肿瘤坏死因子(TNF)-α诱导内皮细胞抗凋亡的机制。方法:在原代培养的牛主动脉内皮细胞(BAECs)中,分别转染细胞色素P450表氧化酶基因rAAV-F87V2周,证实其在内皮细胞中高效表达。用TNF-α(10ng/ml)和放线菌素D(ActD)(5ng/ml)诱导凋亡,流式细胞计数观察其凋亡变化,用Western blot法检测蛋白激酶B(AKT)及细胞外信号调节蛋白激酶(ERK)的磷酸化水平,用流式细胞计数的方法观察空白组及转染F87V组加入AKT抑制剂、LY294002、芹菜素及PD98059后的细胞凋亡比例。结果:与对照组比较,转染细胞色素P450表氧化酶基因后BAECs能高效表达P450表氧化酶基因F87V(P<0.05);流式计数凋亡细胞数,转染表氧化酶较对照组明显减少(P<0.05);转染CYP P450表氧化酶后,AKT及ERK的磷酸化增加;磷脂酰肌醇3激酶(PI3K)、丝裂原活化蛋白激酶(MAPK)和MKK的抑制剂后转染F87V组较对照组凋亡细胞比例明显减少(P<0.05)。结论:细胞色素P450表氧化酶基因rAAV-F87V能通过MAPK(ERK1/2)与PI3K/AKT途径发挥抗凋亡作用。

Protect effect of CYP450 against endothelial cells apoptosis via MAPK and P13K/AKT signaling pathways

Objective:To investigate the protect effect of arachidonic acids cytochrome P450 expoxygenases against endothelial cells apoptosis induced by tumor necrosis factor(TNF)-α. Method:Arachidonic acids cytochrome P450 expoxygenases genes CYPF87V,which had been constructed in recombinant adeno-associated viral expression vectors(rAAV),were transfected into cultured bovine aortic endothelial cells(BAECs).Two weeks later,BAECs were treated with TNF-α(10 ng/ml) and ActD(5 ng/ml).Apoptotic responses were assessed by flow cytometry assay.The level of phosphorylation of AKT and ERK were detected by Western blot.Apoptotic responses were assessed by flow cytometry assay after the addition of AKT inhibitor,LY294002,apigenin,PD98059. Result:The apoptotic rate of the group transfected by CYPF87V was lower than that of the control group.CYPF87V enhanced the phosphorylation of AKT and ERK.After the addition of AKT Inhibitor,LY294002,apigenin,PD98059,the apoptotic rate of CYPF87V group was lower than that of the control group. Conclusion:Expoxygenase could reduce the apoptosis of endothelial cells mediated by the activation of ERK1/2 and PI3K/Akt signaling pathways.