花生四烯酸细胞色素P450表氧化酶代谢产物EET抗脂多糖诱导的内皮细胞凋亡的保护作用及作用机制的研究
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摘要
研究背景:表氧化二十碳三烯酸(Epoxyeicosatrienoic Acids, EETs)是花生四烯酸(Arachidonic Acid,AA)经细胞色素P450(Cytochrome P450 , CYP450)表氧化酶代谢产生的生物活性物质。AA是人体内多种重要心血管活性物质的前体,主要是以酯化的形式存在于细胞膜的内表面,在脂解激素作用下可由磷脂酶A2水解释放至细胞浆内。对AA的研究已有多年,最为人们熟知的是环氧化酶和脂质氧化酶代谢途径,近年研究发现还可通过细胞色素P450途径代谢(即AA代谢的第三条途径),AA经表氧化酶代谢产生四种EETs (5,6-, 8,9-, 11,12-和14,15-EET)。EETs具有与一氧化氮(NO)和前列环素(PGI2)相似的舒张血管的特性,由于早期研究发现它通过激活钙离子敏感的钾通道,使平滑肌细胞处于超极化状态而扩张血管,因此被提议为内皮源性超极化因子(Endothelium-Derived Hyperpolaring Factors , EDHFs)。近几年的研究发现,EETs在脉管系统中发挥着扩张血管以外的多种重要生物学作用,例如,生理量的EETs(100nM)或过度表达CYP-2J2可以减少细胞因子诱导的内皮细胞粘附分子的表达。国外已有学者研究发现EETs具有抗过氧化氢、鬼臼乙叉苷等因素诱导LLCPKcl4细胞凋亡的作用,然而EETs是否也具有抑制LPS诱导牛主动脉内皮细胞的凋亡作用,尚无相关报道。本研究通过给予BAECs外源性的EETs或重组腺相关病毒(recombinant adeno-associated virus,rAAV)介导的表氧化酶基因过度表达,研究它们对LPS诱导BAECs凋亡的影响,同时对其相关的信号转导机制进行初步探讨。
方法:在原代培养的BAECs中,分别给予三种外源性EETs(8,9-, 11,12-,和14,15-EET)一小时后或利用重组腺相关病毒转染细胞色素P450表氧化酶基因CYP-2C11OR, CYP-2J2和CYP-F87V一周后,用LPS(100ng/ml)诱导内皮细胞凋亡,通过细胞形态学(吖啶橙/溴化乙锭染色)、Caspase3活性检测和流式细胞仪计数观察其凋亡变化,同时用Western blot法检测抗凋亡蛋白Bcl-2的表达水平,以及信号分子PI-3K/AKT和丝裂原活化蛋白激酶(MAPK)P38、ERK1/2的磷酸化水平。分别用MEK抑制剂PD 98059、MAPK抑制剂Apigenin、PI-3K抑制剂LY 294002和AKT抑制剂预处理细胞后,观察EETs对LPS诱导的BAECs凋亡的影响。
结果:LPS对BAECs的毒性作用呈剂量依赖性和时间依赖性。LPS可明显诱导BAECs凋亡,但8,9-, 11,12-,和14,15-EET干预的BAECs凋亡细胞数(分别为37.03%±3.014%、33.45%±7.918%和35.75%±7.858% )明显少于溶媒对照组(47.33%±3.154%)。同样地,转染细胞色素P450表氧化酶基因后,由LPS诱导的凋亡细胞数明显少于对照组。而内源性P450表氧化酶抑制剂17-ODYA处理组的凋亡细胞数明显高于对照组。EET处理组和转基因组BAECs的Bcl-2和PI-3K表达水平,ERK1/2和AKT的磷酸化水平均高于对照组,P38磷酸化水平低于对照组。而17-ODYA处理组亦与之相反。MEK抑制剂PD 98059、MAPK抑制剂Apigenin、PI-3K抑制剂LY 294002和AKT抑制剂均可部分阻断EETs的作用。
结论:细胞色素P450表氧化酶代谢产物EETs能明显的抑制LPS诱导的BAECs凋亡,其机制可能是EETs通过下调P38的磷酸化水平,增加ERK的磷酸化水平,减缓Bcl-2的降解,以及激活PI-3K/AKT和其他多条途径发挥抗凋亡作用。这表明细胞色素P450表氧化酶可能有保护心血管系统,防止其受到炎症损伤的作用。
Background:The epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP) epoxygenase arachidonic acid (AA) metabolites. AA is present in vivo in the form esterified to glycerophospholipids inside of cell membrane. Activation of phospholipases (e.g. cytosolic phospholipase A2) releases free AA from the phospholipid (PL) pools and makes it available for oxidative metabolism by several enzyme systems. The PGHSs and LOXs have been extensively studied, and the“third pathway”of AA metabolism was found, wherein multiple cytochrome P450 epoxygenases metabolize AA to four epoxyeicosatrienoic acid regiosomes: 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET. EETs have vasodilatory properties similar to that of nitrogen monoxidum(NO)and prostacyclin(PGI2). Early studies have demonstrated that EETs could make smooth muscle cell (SMC) hyperpolarized through opening Ca2+-sensitive potassium channel(KCa2+), so EETs was proposed to be endothelial derived hyperpolarizing factors (EDHFs). EETs have recently been shown to play important nonvasodilatory roles within the vasculature, for example, physiological concentrations (100nM) of EETs or overexpression of human CYP2J2 decreased cytokine-induced endothelial cell adhesion molecule expression. In recent years, mang studies have already shown that EETs could inhibit LLCPKcl4 cell apoptosis induced by H2O2, etoposide etc. However, it remains elucidated whether EETs can protect bovine aortic endothelial cells (BAECs) against apoptosis induced by LPS. In this study, therefore, we investigated the effects of endogenous and exogenous EETs on apoptosis of BAECs induced by LPS and the relevant signaling mechanisms involved.
Methods: CYP expoxygenase genes, CYP2C11OR, CYP2J2 and CYPF87V were transfected into cultured BAECs for 1 weeks using recombinant adeno-associated viral vector (rAAV), or EETs were added into the culture medium directly. BAECs were treated with LPS (100 ng/ml) or pretreated with mitogenactivated protein kinase kinase inhibitor PD98059, mitogenactivated protein kinase inhibitor Apigenin, phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 and inhibitor of AKT. Apoptosis was detected by morphological observations (Acridine Orange/Ethidium Bromide staining), flow cytometry assay and Caspase3 activity assay. As well the level of bcl-2, PI-3K, phospho-Akt, mitogen-activated protein kinase (MAPK) phospho-P38 and the phospho-ERK1/2 were probed using corresponding antibodies by Western blots. Results: LPS treatment significantly induced BAEC apoptosis, but incubation with 8,9-, 11,12-, and 14,15-EET markedly attenuated LPS-induced BAEC apoptosis (37.03%±3.014%, 33.45%±7.918% and 35.75%±7.858%, respectively) compared with vehicle control (47.33%±3.154%). Also CYP epoxygenase genes attenuated LPS-induced BAEC apoptosis and significantly reduced the ratios of apoptic BAECs. Moreover, addition of CYP epoxygenase inhibitor 17-ODYA reversed antiapoptic effects of epoxygenase gene overexpressions. Furthermore, in BAECs pretreated with EET or transfected with cytochrome P450 epoxygenase, the expression of Bcl-2, PI-3K, phosphorylation of ERK1/2, phosphorylation of Akt were significantly increased compared with that of controls, in contrast, phosphorylation of P38 were less. The antiapoptotic effects of EETs were partially blocked by mitogen activated protein kinase kinase inhibitor PD98059, mitogenactivated protein kinase inhibitor Apigenin, PI-3K inhibitor LY294002 and inhibitor of Akt.
Conclusion: LPS induced apoptosis of the cultured endothelial cells and EETs as well as forced overexpression of epoxygenase genes can inhibits LPS-induced endothelial apoptosis partially by activation of MAPK and PI-3K/Akt pathways, which may reduce degradation of bcl-2 and activation of caspase-3. These results suggest that CYP epoxygenases may protect cardiovascular system from inflammatory injury and atherosclerosis.
方法:在原代培养的BAECs中,分别给予三种外源性EETs(8,9-, 11,12-,和14,15-EET)一小时后或利用重组腺相关病毒转染细胞色素P450表氧化酶基因CYP-2C11OR, CYP-2J2和CYP-F87V一周后,用LPS(100ng/ml)诱导内皮细胞凋亡,通过细胞形态学(吖啶橙/溴化乙锭染色)、Caspase3活性检测和流式细胞仪计数观察其凋亡变化,同时用Western blot法检测抗凋亡蛋白Bcl-2的表达水平,以及信号分子PI-3K/AKT和丝裂原活化蛋白激酶(MAPK)P38、ERK1/2的磷酸化水平。分别用MEK抑制剂PD 98059、MAPK抑制剂Apigenin、PI-3K抑制剂LY 294002和AKT抑制剂预处理细胞后,观察EETs对LPS诱导的BAECs凋亡的影响。
结果:LPS对BAECs的毒性作用呈剂量依赖性和时间依赖性。LPS可明显诱导BAECs凋亡,但8,9-, 11,12-,和14,15-EET干预的BAECs凋亡细胞数(分别为37.03%±3.014%、33.45%±7.918%和35.75%±7.858% )明显少于溶媒对照组(47.33%±3.154%)。同样地,转染细胞色素P450表氧化酶基因后,由LPS诱导的凋亡细胞数明显少于对照组。而内源性P450表氧化酶抑制剂17-ODYA处理组的凋亡细胞数明显高于对照组。EET处理组和转基因组BAECs的Bcl-2和PI-3K表达水平,ERK1/2和AKT的磷酸化水平均高于对照组,P38磷酸化水平低于对照组。而17-ODYA处理组亦与之相反。MEK抑制剂PD 98059、MAPK抑制剂Apigenin、PI-3K抑制剂LY 294002和AKT抑制剂均可部分阻断EETs的作用。
结论:细胞色素P450表氧化酶代谢产物EETs能明显的抑制LPS诱导的BAECs凋亡,其机制可能是EETs通过下调P38的磷酸化水平,增加ERK的磷酸化水平,减缓Bcl-2的降解,以及激活PI-3K/AKT和其他多条途径发挥抗凋亡作用。这表明细胞色素P450表氧化酶可能有保护心血管系统,防止其受到炎症损伤的作用。
Background:The epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP) epoxygenase arachidonic acid (AA) metabolites. AA is present in vivo in the form esterified to glycerophospholipids inside of cell membrane. Activation of phospholipases (e.g. cytosolic phospholipase A2) releases free AA from the phospholipid (PL) pools and makes it available for oxidative metabolism by several enzyme systems. The PGHSs and LOXs have been extensively studied, and the“third pathway”of AA metabolism was found, wherein multiple cytochrome P450 epoxygenases metabolize AA to four epoxyeicosatrienoic acid regiosomes: 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET. EETs have vasodilatory properties similar to that of nitrogen monoxidum(NO)and prostacyclin(PGI2). Early studies have demonstrated that EETs could make smooth muscle cell (SMC) hyperpolarized through opening Ca2+-sensitive potassium channel(KCa2+), so EETs was proposed to be endothelial derived hyperpolarizing factors (EDHFs). EETs have recently been shown to play important nonvasodilatory roles within the vasculature, for example, physiological concentrations (100nM) of EETs or overexpression of human CYP2J2 decreased cytokine-induced endothelial cell adhesion molecule expression. In recent years, mang studies have already shown that EETs could inhibit LLCPKcl4 cell apoptosis induced by H2O2, etoposide etc. However, it remains elucidated whether EETs can protect bovine aortic endothelial cells (BAECs) against apoptosis induced by LPS. In this study, therefore, we investigated the effects of endogenous and exogenous EETs on apoptosis of BAECs induced by LPS and the relevant signaling mechanisms involved.
Methods: CYP expoxygenase genes, CYP2C11OR, CYP2J2 and CYPF87V were transfected into cultured BAECs for 1 weeks using recombinant adeno-associated viral vector (rAAV), or EETs were added into the culture medium directly. BAECs were treated with LPS (100 ng/ml) or pretreated with mitogenactivated protein kinase kinase inhibitor PD98059, mitogenactivated protein kinase inhibitor Apigenin, phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 and inhibitor of AKT. Apoptosis was detected by morphological observations (Acridine Orange/Ethidium Bromide staining), flow cytometry assay and Caspase3 activity assay. As well the level of bcl-2, PI-3K, phospho-Akt, mitogen-activated protein kinase (MAPK) phospho-P38 and the phospho-ERK1/2 were probed using corresponding antibodies by Western blots. Results: LPS treatment significantly induced BAEC apoptosis, but incubation with 8,9-, 11,12-, and 14,15-EET markedly attenuated LPS-induced BAEC apoptosis (37.03%±3.014%, 33.45%±7.918% and 35.75%±7.858%, respectively) compared with vehicle control (47.33%±3.154%). Also CYP epoxygenase genes attenuated LPS-induced BAEC apoptosis and significantly reduced the ratios of apoptic BAECs. Moreover, addition of CYP epoxygenase inhibitor 17-ODYA reversed antiapoptic effects of epoxygenase gene overexpressions. Furthermore, in BAECs pretreated with EET or transfected with cytochrome P450 epoxygenase, the expression of Bcl-2, PI-3K, phosphorylation of ERK1/2, phosphorylation of Akt were significantly increased compared with that of controls, in contrast, phosphorylation of P38 were less. The antiapoptotic effects of EETs were partially blocked by mitogen activated protein kinase kinase inhibitor PD98059, mitogenactivated protein kinase inhibitor Apigenin, PI-3K inhibitor LY294002 and inhibitor of Akt.
Conclusion: LPS induced apoptosis of the cultured endothelial cells and EETs as well as forced overexpression of epoxygenase genes can inhibits LPS-induced endothelial apoptosis partially by activation of MAPK and PI-3K/Akt pathways, which may reduce degradation of bcl-2 and activation of caspase-3. These results suggest that CYP epoxygenases may protect cardiovascular system from inflammatory injury and atherosclerosis.
引文
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