17β-雌二醇诱导血管内皮细胞释放一氧化氮的作用机制∗

摘要

Objective To investigate the time and dose dependent effects of 17 β-estradiol ( E2 ) on eNOS phosphorylation and nitric oxide (NO) production from bovine aortic endothelial cells (BAECs). Methods The BAECs were cultured in 24-well flat plate.The dose dependent rapid induction of NO release by E2 in the BAECs was explored,and the non-genomic mechanism was studied. Each experiment was repeated for 3 times. The NO level was quantified by the electron spin resonance spectroscopy(ESR)method,and the phosphorylation of eNOS were determined by Western blot. Results NO release increased time dependently from BAECs after treatment with E2 at 100 nmol•L-1 at 1,5,10 and 15 min,and the effect peaked at 10 min.There were dose dependent effects of NO production as well as eNOS phosphorylation after treatment with E2 at different concentrations for 10 min,and the effect was the most obvious at the concentration of 100 nmol•L-1 .Upon treatment with equal volume of saline,E2 and actinomycin D (25 μg•mL-1 ) for 10 min,the NO release was(5.38±2.35),(10.59±3.28)and(10.68± 3.31) nmol•mg-1 ,respectively,and the eNOS phosphorylation level was 0.36±0.03,0.98±0.08 and 0.99±0.08,respectively. Compared with 0.9% sodium chloride solution,the NO release and eNOS phosphorylation were significantly increased in E2 treated cells(all P<0.05). Conclusion 17 β-estradiol at 100 nmol•L-1 induced eNOS phosphorylation as well as NO production from bovine vascular endothelial cells through non-genomic mechanism.The effect peaked at 10 minutes.%目的：观察17β-雌二醇(E2)诱导小牛胸主动脉内皮细胞(BAECs)快速激活并释放一氧化氮(NO)的时间和浓度依赖性趋势。方法采用24孔平底细胞培养板培养 BAECs 分别进行实验,观察不同浓度 E2诱导 BAECs 快速激活并释放 NO 的浓度依赖性趋势及非基因组机制通路。每次实验均重复3次。使用电子自旋共振波谱法定量测定 NO 的释放；蛋白免疫印迹法检测 BAECs 一氧化氮合成酶(eNOS)磷酸化的水平。结果终浓度为100 nmol•L－1的 E2分别处理 BAECs 1,5,10和15 min 后,NO 的释放量呈现时间依赖性,在10 min 达到高峰；培养液中加入不同终浓度的 E2分别处理 BAECs 10 min后,NO 的释放量和 eNOS 的磷酸化均显示有剂量依赖性,并在终浓度为100 nmol•L－1时达到高峰；培养液中分别加入等体积0.9％氯化钠溶液、100 nmol•L－1 E2和25μg•mL－1放线菌素 D(Act-D)处理 BAECs 10 min 后,NO 释放量分别为(5.38±2.35),(10.59±3.28)和(10.68±3.31) nmol•mg－1；eNOS 的磷酸化水平分别为0.36±0.03,0.98±0.08和0.99±0.08；与经0.9％氯化钠溶液处理比较,经100 nmol•L－1 E2处理后,BAECs 的 NO 释放量和 eNOS 的磷酸化水平均显著增加(均 P<0.05)。结论终浓度为100 nmol•L－1 E2可以通过非基因组机制快速激活血管内皮细胞中 eNOS 的磷酸化水平,进而可以快速合成和释放 NO 发挥其生物学作用,10 min 即可使此效应达到峰值。