17β-雌二醇诱导血管内皮细胞释放一氧化氮的作用机制

Mechanism of 17β-estradiol Induced Nitric Oxide Release from Bovine Aortic Endothelial Cells

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(e NOS)磷酸化的水平。结果终浓度为100 nmol·L-1的E2分别处理BAECs1,5,10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BAECs10 min后,NO的释放量和e NOS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-1E2和25μg·m L-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;e NOS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-1E2处理后,BAECs的NO释放量和e NOS的磷酸化水平均显著增加(均P<0.05)。结论终浓度为100 nmol·L-1E2可以通过非基因组机制快速激活血管内皮细胞中e NOS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。

Objective To investigate the time and dose dependent effects of 17 β-estradiol( E2) on e NOS 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 nongenomic 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 e NOS were determined by Western blot. Results NO release increased time dependently from BAECs after treatment with E2 at 100 nmol·L-1at 1,5,10 and 15 min,and the effect peaked at10 min.There were dose dependent effects of NO production as well as e NOS 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·m L-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 e NOS 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 e NOS phosphorylation were significantly increased in E2 treated cells( all P< 0. 05). Conclusion 17 β-estradiol at 100 nmol · L-1induced e NOS phosphorylation as well as NO production from bovine vascular endothelial cells through non-genomic mechanism.The effect peaked at 10 minutes.