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目的观察PPARα激动剂非诺贝特对牛主动脉(BAECs)内皮细胞一氧化氮合酶(eNOS)活性和表达的影响。方法制备5-9代BAECs,加入不同浓度的非诺贝特(0,5,10,50,100μmol/L)后,用NOSAssayKit测定eNOS活性,RT-PCR法检测eNOSmRNA表达,Westernblot分析检测eNOS蛋白质表达。结果非诺贝特以浓度和时间依赖的方式增加eNOS活性,非诺贝特浓度10μmol/L以上时,明显增加eNOS活性。50μmol/L非诺贝特处理48h时eNOS活性最大(为对照组的232±047倍,P<001)。非诺贝特处理1h和12h不增加eNOS活性。RT-PCR分析表明,非诺贝特浓度大于5μmol/L以上时,明显增加eNOSmRNA水平,在非诺贝特浓度为50μmol/L时作用最大,为对照组的208±033倍(P<001)。此作用在6h时出现,持续到48h。Westernblot显示,非诺贝特处理48h,eNOS蛋白表达明显增加,在浓度为10,50和100μmol/L时,eNOS蛋白表达分别为对照组的180±045,270±042和220±032倍,均P<001。在非诺贝特处理12h后出现,持续到48h。结论PPARα激动剂非诺贝特增加BAECseNOS基因表达,提高eNOS活性及增加蛋白表达。
Objective To observe the effects of fenofibrate, a PPARα agonist, on the activity and expression of nitric oxide synthase (eNOS) in endothelial cells of bovine aortic (BAECs). Methods 5-9 passages of BAECs were prepared. After adding different concentrations of fenofibrate (0, 5, 10, 50 and 100μmol / L), eNOS activity was determined by NOSAssay Kit, eNOS mRNA expression was detected by RT-PCR and eNOS protein by Western blot analysis . Results Fenofibrate increased eNOS activity in a concentration- and time-dependent manner. When fenofibrate concentration was above 10 μmol / L, eNOS activity was significantly increased. The eNOS activity was maximal 48 h after treatment with 50 μmol / L fenofibrate (232 ± 0.47 fold, P <001) of the control group. Fenofibrate did not increase eNOS activity at 1 and 12 h. RT-PCR analysis showed that when fenofibrate was more than 5μmol / L, the level of eNOS mRNA was significantly increased, which was the highest at fenofibrate concentration of 50μmol / L, which was 208 ± 033 times of the control group (P <001) . This effect occurs at 6h and lasts up to 48h. Western blot showed that eNOS protein expression increased significantly after fenofibrate treatment for 48h, and eNOS protein expression was 180 ± 045, 270 ± 042 and 220 ± 032 times of the control group at 10, 50 and 100μmol / L, respectively, P < 001. Appeared after fenofibrate treatment for 12h and lasted 48h. Conclusion PPARα agonist fenofibrate increases BAECseNOS gene expression, increases eNOS activity and increases protein expression.