花生四烯酸细胞色素P450表氧化酶对凋亡内皮细胞Bcl-2表达、Caspase-3及MAPK活性的影响

Effect of Arachidonic Acid Cytochrome P450 Epoxygenase on Bcl-2 Expression, Capase-3 and MAPK Activity in Apoptotic Endothelial Cells Induced by TNF-α

花生四烯酸经细胞色素P450表氧化酶代谢产生的内皮来源超极化因子(EDHF)[表氧化二十烷烯酸(EETs)]对内皮细胞具有保护作用。研究了转染细胞色素P450表氧化酶基因CYPBM3·F87V、CYP2C11OR及CYP2J2产生内源性EETs,通过检测内皮细胞中Bcl-2表达、caspase-3的活性及MAPK磷酸化水平探讨内源性EDHF的内皮细胞保护效应及其抗TNF-α诱导内皮细胞凋亡的作用机制。原代培养的牛主动脉血管内皮细胞转染CYP450表氧化酶基因24h后,加入TNF-α作用一定时间诱导内皮细胞凋亡,用Western印迹方法检测Bcl-2的表达,MAPK磷酸化水平,同时测定caspase-3的活性。结果显示转染表氧化酶基因能抑制TNF-α诱导的时间依赖性Bcl-2下调,抑制Caspase-3的激活。TNF-α使细胞内磷酸化MAPK水平呈时间依赖性减低,转染表氧化酶基因后细胞内的磷酸化MAPK水平较对照组升高。因此,转染表氧化酶基因CYPBM3·F87V、CYP2C11OR以及CYP2J2使内皮细胞产生内源性EETs(EDHF)通过激活MAPK(ERK1/2)途径,抑制抗凋亡基因Bcl-2的降解,抑制caspase-3的激活,从而抑制TNF-α诱导的内皮细胞凋亡,因而具有内皮保护效应。

Endothelium-derived hyperpolarizing factor （EHDF） may protect endothelial cells against apoptosis induced by TNF-α, the purpose of the present study was to investigate the effects of endogenous EDHF produced by cytochrome P450 （CYP） epoxygenases transfection on Bcl-2 levels, capase-3 activity and the phosphorylation of MAPK in apoptotic endothelial cells induced by TNF-α. Three or four passages of cultured bovine aortic endothelial cells （BAECs） were transfected with CYPBM3·F87V, CYP2C11OR, CYP2J2 or the empty vector （pCB6）. Twenty-four hours later transfected cells were incubated with TNF-a for appropriate time, the Bcl-2 levels and MAPK phosphorylation were detected by Western blot analysis, and capase-3 activity was examined by using DEVD-p-nitroanilide as a substrate. TNF-α decreased Bcl-2 protein level in a time-dependent manner and increased significantly caspase-3 activity. Transfection of BAECs with epoxygenases prevented the decrease of Bcl-2 protein and caspase-3 activation induced by TNF-α compared with controls. Incubation of BAECs with TNF-α induced a marked dephosphorylation of MAPK in a time-dependent manner. Transfection with epoxygenases increased the phosphorylation levels of MAPK at all examined time points. The present study demonstrates that transfection of CYP epoxygenases may protect endothelial cells and inhibit endothelial apoptosis by increasing the phosphorylation level of ERK1/2, preventing both degradation of anti-apoptotic protein Bcl-2 and activation of caspase-3 induced by TNF-α.