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目的研究过氧化物酶体增生物激活受体α(peroxisomeproliferator-activatedreceptoralpha,PPARα)的配体苯扎贝特对原代牛主动脉内皮细胞(bovineaortaendothelialcells,BAEC)一氧化氮合酶(endothelialnitricoxidesynthase,eNOS)基因表达的影响并探讨其机制。方法分离和培养牛主动脉内皮细胞,采用Northern印迹法、Western印迹法检测苯扎贝特对BAECeNOSmRNA和蛋白质表达的影响,采用定量PCR的方法及NO试剂盒检测苯扎贝特对eNOSmRNA半衰期及NO产生的影响;继而采用Western印迹法,给予不同的信号转导通路抑制剂研究苯扎贝特影响eNOS表达所通过的信号转导途径,此外,构建了由人eNOS启动子驱动的荧光报告基因,研究苯扎贝特对eNOS启动子活性的影响。结果苯扎贝特以浓度(50~200μmol/L)依赖的方式明显上调BAEC细胞eNOS的mRNA和蛋白质表达(P<0.05),并促进一氧化氮(nitricoxide,NO)的生成[对照组(14.97±1.29)μmol/L,苯扎贝特不同浓度组(25.12±1.25)μmol/L,(30.12±1.85)μmol/L,(33.47±1.22)μmol/L],增强eNOS-ser-1179位点的磷酸化表达(P<0.05),但是对eNOS-thr-497位点的磷酸化表达几乎没有抑制作用,定量PCR证实苯扎贝特增加eNOSmRNA的半衰期(从3.1~6.1h),进一步的研究显示苯扎贝特以浓度依赖的方式增加人eNOS启动子驱动的荧光报告基因的荧光活性(相对的荧光活性在100μmol/L和200μmol/L组分别为4429.43±391.41,6187.5±307.53,对照组为3361.81±316.85),增加磷酸化丝裂原激活的蛋白激酶(mitogen-activatedproteinkinase,MAPK)的蛋白质表达(P<0.05及P<0.01),而PPARα、磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase,PI3K)和MAPK抑制剂可明显逆转苯扎贝特对eNOS表达的上调作用(P<0.01)。结论苯扎贝特通过上调eNOS的蛋白质表达、促进eNOS的磷酸化、增强eNOS的转录及eNOSmRNA的稳定性,从而促进NO的生成,其效应的发挥既通过依赖于PPARα的方式,也可以经MAPK和PI3K信号通路介导的不依赖于PPARα的“非基因效应”,揭示了PPARα的配基苯扎贝特的降脂外作用包括抗动脉粥样硬化和抗高血压的可能作用机制。
Objective To investigate the effect of bezafibrate, a ligand of peroxisomeproliferator-activated receptor (PPARα), on the expression of endothelial nitric oxide synthase (eNOS) in primary bovine cordurgery endothelial cells (BAEC) Gene expression and explore its mechanism. Methods The endothelial cells of bovine aorta were isolated and cultured. The effects of bezafibrate on the expression of BAECeNOS mRNA and protein were detected by Northern blotting and Western blotting. The benazepan etanercept assay was used to detect the half-life and NO And then using Western blotting method to give different signal transduction pathway inhibitors to study bezafibrate eNOS expression through the signal transduction pathway, in addition, the construction of a human eNOS promoter-driven fluorescent reporter gene, Effects of bezafibrate on eNOS promoter activity. Results Bezafibrate significantly increased the mRNA and protein expression of eNOS in BAEC cells in a concentration dependent manner (50 ~ 200μmol / L), and promoted the formation of nitric oxide (NO) [control group (14.97 Serum levels of eNOS-ser-1179 were significantly increased in the different concentrations of bezafibrate (25.12 ± 1.25) μmol / L, (30.12 ± 1.85) μmol / L and (33.47 ± 1.22) μmol / (P <0.05), but had little inhibitory effect on the phosphorylation of eNOS-thr-497. Quantitative PCR confirmed that bezafibrate increased the half-life of eNOS mRNA (from 3.1 to 6.1 h), and further studies It was shown that bezafibrate increased the fluorescence activity of the human eNOS promoter-driven fluorescent reporter in a concentration-dependent manner (relative fluorescence activity was 4429.43 ± 391.41,6187.5 ± 307.53 in 100 μmol / L and 200 μmol / L groups, respectively, 3361.81 ± 316.85), increased the protein expression of mitogen-activated protein kinase (MAPK) (P <0.05 and P <0.01), while PPARα, phosphatidylinositol 3-kinase PI3K) and MAPK inhibitor could obviously reverse the up-regulation of eNOS expression by bezafibrate (P <0.01). Conclusion Bezafibrate can promote the production of NO by up-regulating the protein expression of eNOS, promoting the phosphorylation of eNOS, enhancing the transcription of eNOS and the stability of eNOS mRNA. The effect of bezafibrate exerted both PPARα-dependent and MAPK And the PPARα-independent “non-gene effect” mediated by the PI3K signaling pathway revealed that the lipid-lowering effect of PPARα ligand bezafibrate may include the possible mechanism of anti-atherosclerosis and antihypertensive effect.