花生四烯酸表氧化酶抗血管内皮细胞凋亡研究

Arachidonic Acid Cytochrome P450 Epoxygenases Protect Vessel Endothelial Cells Against Apoptosis Induced by Tumor Necrosis Factor ABSTRACT:The vascular endothelium synthesizes and releases several vasodilating factors, including nitric oxide and prostacyclin. These factors play very important role.in regulating vascular tone and cardiovascular homeostasis and are also considered as“protective factors”. Recently another important endothelium-derived relaxing factor has been identified and termed as endothelium-derived hyperpolarizing factor.(EDHF). The epoxyeicosatrienoic acids (EETs), cytochrome P-450.epoxygenase-dependent metabolites of arachidonic acid, have properties similar to those of EDHF and are considered to be EDHF. One of our previous studies has.demonstrated that EDHF significantly up-regulated the endothelial nitric oxide.synthase (eNOS) gene expression and enhanced eNOS activity. In the present study, we investigated whether endogenous EHDFs produced by CYP epoxygenases.transfection were able to protect endothelial cells against apoptosis induced by tumor necrosis factor alpha and elucidated underlying signal pathways involved in. Three or.four passages of cultured bovine aortic endothelial cells (BAECs) were transfected.with CYP BM3F87V, CYP2C11-CYPOR, CYP2J2 or vector (pCB6), and then the.transfected cells were incubated with TNF-α. Overexpression of these CYP450.epoxygenases significantly inhibited TNF-α-induced apoptosis in BAEC as evaluated.by cell viability assay, DNA ladder analysis, flow cytometry assay and morphologicalobservations under fluorescence microscopy. TNF-α significantly decreased Bcl-2.protein level in a time-dependent manner and increased caspase-3 activity in the.control cells. Transfection of BAECs with epoxygenases prevented the Bcl-2 protein.degradation and caspase-3 activation induced by TNF-α. The effect of TNF-α on.MAPK phosphorylation was also detected by Western blot analysis with a.phosphospecific MAPK antibody. 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 time points.examined. The present study demonstrates that overexpression of CYP epoxygenasesprotect endothelial cells against TNF-α-induced apoptosis. The antiapoptotic effect ofCYP epoxygenases were mediated by increasing the phosphorylation level of ERK,preventing TNF-α–stimulated anti-apoptotic protein Bcl-2 degradation and caspase-3activity in endothelial cells.

血管内皮细胞损伤是心脑血管病发生和发展的核心环节。本项目在新鲜分离培养的牛主动脉内皮细胞中转染花生四烯酸表氧化酶基因（在内皮细胞产生EDHF），加凋亡诱导剂后通过细胞形态观察，流式细胞仪分析和Bc1-2及Caspases系统分析判断细胞凋亡状态并研究其机理，从而探索内皮超极化因子抗内皮细胞损伤和凋亡作用，尾心血管病防治研究提供理论依据。.