非诺贝特上调血管内皮细胞一氧化氮合酶的表达

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, 100 μmol/L)后,用NOS Assay Kit测定eNOS活性,RT-PCR法检测eNOS mRNA表达,Western blot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10 μmol/L以上时,明显增加eNOS活性。50μmol/L非诺贝特处理48 h时eNOS活性最大(为对照组的2.32±0.47倍,P<0.01)。非诺贝特处理1 h和12 h不增加eNOS活性。RT-PCR分析表明,非诺贝特浓度大于5 μmol/L以上时,明显增加eNOS mRNA水平,在非诺贝特浓度为50 μmol/L时作用最大,为对照组的2.08±0.33倍(P<0.01)。此作用在6 h时出现,持续到48 h。Western blot显示,非诺贝特处理48 h,eNOS蛋白表达明显增加,在浓度为10,50 和100 μmol/L时,eNOS蛋白表达分别为对照组的1.80±0.45, 2.70±0.42 和 2.20±0.32 倍,均P<0.01。在非诺贝特处理12 h后出现,持续到48 h。结论:PPARα激动剂非诺贝特增加BAECs eNOS基因表达,提高eNOS活性及增加蛋白表达。

Fenofibrate increases endothelial nitric oxide synthase expression in bovine aortic endothelial cells

AIM: We hypothesize that peroxisome proliferator- activated receptor α(PPARα) agonists act directly on nitric oxide (NO) production in vascular endotheliun. Thus, the purpose of this study is to investigate the effects of fenofibrate on endothelial NO synthase(eNOS) activity and its expression in cultured vascular endothelial cells.METHODS: Bovine aortic endothelial cells (BAECs) were treated with the PPARα activator fenofibrate. The eNOS activity and the expression of eNOS protein and its mRNA were determined. RESULTS: Our data show that fenofibrate increased eNOS activity in a dose - and time - dependent manner. At the concentration of 10 μnol/L or more, fenofibrate treatment caused a significant increase in eNOS activity. The maximal increase in eNOS activity(2.32 + 0.47 fold of the control) was observed with 50 μmol/L fenofibrate treatment for 48 h. Fenofibrate failed to increase eNOS activity at 1 and12 h. RT- PCR analysis demonstrated that eNOS mRNA relative to β - actin mRNA significantly increased at concentrations of 5 μmol/L or more. It reached 2.08 + 0.33 fold of the control with 50 μmol/L fenofibrate. Significant increase in eNOS mRNA levels was observed after 6 h, and lasted for 48 h. The peak increase in eNOS mRNA levels(2.13 + 0.30fold of the control, P ＜ 0.01 ) was observed with 50 μmol/L fenofibrate treatment for 12 h. Longer incubation of cells with 50 μmol/L fenofibrate caused no further increase. The treatment of BAECs with fenofibrate for 48 h demonstrated a concentration- dependent increase in eNOS protein levels as measured by Western blot analysis. Densitometrc analysis indicated that there was a significant increase in eNOS to β- actin ratios after fenofibrate treatment at concentrations of10,50 and 100 μmol/L(1.80 + 0.45, 2.70+0.42 and 2.20+0.32 fold of the control, respectively, P ＜0.01). The significant increase in eNOS protein levels was observed 12 h after treatment and lasted for 48 h. CONCLUSION:PPARα activator, fenofibrate, enhances endothelial NO production by directly upregulating eNOS expression and activity.