非诺贝特通过上调三磷酸鸟苷环化水解酶Ⅰ促进eNOS复偶联

摘要

Objective To investigate whether fenofibrate could re-couple endothelial nitric oxide synthase (eNOS) through upregulating the level of Guanosine 5'-triphosphate cyclohydrolase- I (GTPCH- I ) and reversing low-expression of intracellular tetrahydrobiopterin(BH4) in endothelial cells from human umbilical cord vein. Methods Human umbilical vein endothelial cells ( HUVECs) were treated with fenofibrate for 2 hours, after that the cells were incubated with lipopo-lysaccharide (LPS) for 24 hours. The amount of intracellular BH4 was measured by high-performance liquid chromatogra-phy. The level of cell supernatant NO was measured by ELJSA kits. A confocal laser scanning microscope ( Leica) was used to detect the level of intracellular ROS. Western blot was used to detect the level of intracellular GTPCH- I . Results After HUVECs were treated with LPS for 24 hours, cellular levels of BH4 and cell supernatant NO were significantly reduced compared to control group (both P < 0.05), respectively. And the fluorescence intensity of intracellular ROS was significantly increased (P < 0. 05) compared with control group. But pretreated with fenofibrate for 2 hours before cells were induced by LPS, levels of BH4 and cell supernatant NO were significantly raised compared with LPS treatment (both P<0.05). Furthermore, the fluorescence intensity was significantly reduced in fenofibrate group than LPS group (P < 0.05 ). The level of intracellular GTPCH- I was increased dependent of concentration after treated with fenofibrate alone. The expression of GTPCH- I up-regulated by fenofibrate reached the peak at 12 hours. Conclusions Fenofibrate may help protect against atherosclerosis by promoting the re-coupling of eNOS with increased levels of NO and BH4 and decreased level of ROS through increasing the level of intracellular GTPCH-1 .%目的 探讨非诺贝特发挥降脂外血管内皮保护作用的机制.方法 体外培养人脐静脉内皮细胞( HUVECs),非诺贝特预处理HUVECs 2 h,再与脂多糖(LPS)共孵育24h.采用Western blot检测三磷酸鸟苷环化水解酶Ⅰ (GTPCH-Ⅰ)的表达水平,高效液相色谱法检测四氢生物蝶呤(BH4)的表达水平,ELISA检测细胞上清一氧化氮(N0)浓度,利用Confocal方法检测细胞活性氧(ROS)产生水平.结果 非诺贝特预处理后,较单纯LPS刺激组,细胞内BH4表达水平及细胞上清NO产生增多,伴有细胞内ROS产生减少(P均＜0.05)；此外,单独给予非诺贝特处理内皮细胞后,可见浓度依赖性上调细胞GTPCH-Ⅰ的表达,非诺贝特(10 μmol/L)上调GTPCH-Ⅰ的表达在12h达到高峰.结论 非诺贝特通过上调内皮细胞GTPCH-Ⅰ的表达而增加BH4的水平,进而促进内皮型一氧化氮合酶的复偶联,改善血管内皮功能.