苯扎贝特对牛主动脉内皮细胞一氧化氮合酶基因表达的影响及其机制的研究

Bezafibrate up-regulate endothelial nitric oxide gene expressions via peroxisome proliferator-activated receptors alpha-dependent and independent pathways in cultured Bovine endothelial cells

目的 研究过氧化物酶体增生物激活受体α（peroxisome proliferator-activated receptor alpha，PPARα）的配体苯扎贝特对原代牛主动脉内皮细胞（bovine aorta endothelial cells，BAEC）一氧化氮合酶（endothelial nitric oxide synthse，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BAEC eNOS mRNA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eNOS mRNA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（nitric oxide，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-set-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入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-activated protein kinase，MAPK）的蛋白质表达（P〈0．05及P〈0．01），而PPARα、磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，P13K）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PPARa的方式，也可以经MAPK和P13K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 effects and related mechanisms of bezafibrate, a ligand of peroxisome proliferator-activated receptors alpha （ PPARα）, on endothelial nitric oxide synthase （eNOS）. Methods Firstly, in cultured bovine aorta endothelial cells （ BAEC）, the effects of bezafibrate on eNOS mRNA and protein levels were investigated by Northern blot and Western blot. The half-life of eNOS mRNA and NO production determined from BAECs treated with bezafibrate were performed by real-time quantitative PCR and NO assay. Next, the effects of bezafibrate on signal pathways in BAEC, through inhibitors of PPARα, PI3 kinase and MAPK, were investigated by Western blot. Then luciferase reporter gene driven by human eNOS promoter were coloned and transfected endothelial cells to see the effects of bezafibrate on eNOS promoter-driven luciferase activity. Results In cultured BAEC, bezafibrate significantly upregulated eNOS expressions at protein and mRNA levels in a concentration-dependent fashion （50 - 200 μmol/L） （ P 〈 0. 05）, and increased nitric oxide （NO） production, respectively [ control （ 14.97 ± 1.29 ） μmol/L, different concentration groups （25. 12 ± 1.25） μmol/L, （30. 12 ± 1.85） μmol/L, （33.47 ± 1.22） μmol/ L], and enhanced phosphorylation of eNOS-set-liT9 site（P 〈0. 05）, but not eNOS-thr-497 site. Through real-time quantitative PCR, bezafibrate prolonged eNOS mRNA half-life from 3.1 hour to 6. 1 hour were demonstrated. Further studies showed that bezafibrate treatments significantly enhanced dose-dependently luciferase activity （relative luciferase activity in 100 μmol/L and 200 μmol/L groups 4429.43 ±391.41 and 6187.5 ±307. 53）, compared with untreated luciferase reporter gene group （3361.81 ±316. 85） （P〈0. 05 and P 〈 0.01, respectively）, and induced MAPK phosphorylation expression （ P 〈 0. 05 and P 〈 0. 01, respectively）. Then these effects of bezafibate upregulated eNOS expressions could be blocked by PPARα antagonist, MAPK and PI3K inhibitor while not affected by PKC and MEK inhibitor （P 〈 0.01）. Conclusions Bezafibrate can upregulate eNOS expression, enhance eNOS-set-1179 site phosphorylation, increase NO production and stabilize eNOS mRNA through PPARα dependent pathway and PPARct independent MAPK and PI3K pathways. This mechanism provides additional anti-atherosclerotic and anti- hypertension benefits of bezafibrate inaddition of lipid-lowering effects.