血管紧张素-Ⅱ活化肺微血管内皮细胞核因子-κB的研究

目的 探讨血管紧张素Ⅱ(Ang Ⅱ)对肺微血管内皮细胞(HPMEC)核因子-κB(NF-κB)的活性的影响,以及经典途径在Ang Ⅱ介导的NF-κB活化过程中的作用.方法 本实验分为:Ang Ⅱ组:10-6 mol/L AngⅡ分别刺激HPMEC 0、0.5、1、2、4 h;氯沙坦组:10-6mol/L氯沙坦(AngⅡ 1型受体阻滞剂)预先处理HPMEC 1 h,再予相同10-6mol/L AngⅡ刺激细胞2 h,提取胞浆蛋白和胞核蛋白.凝胶电泳迁移率分析实验(EMSA)观察细胞中NF-κB的DNA结合活性.Western印迹检测细胞浆中抑制因子κBα(IκBα)的含量.结果 AngⅡ刺激HPMEC 0.5 h后细胞中NF-κB活性明显上升(144.5±16.1),在2h达到峰值(270.1±27.2),4 h(215.1±17.8)较2 h有所下降,但仍高于0 h水平,以上各时间点与AngⅡ刺激细胞0 h(100.0±25.1)相比,差异有统计学意义(P＜0.05).与Ang Ⅱ刺激HPMEC 0 h IκBα含量(44.4%±2.1%)相比,0.5 h后IκBα含量即开始明显下降(38.9%±3.6%,P＜0.05),2 h后IκBα含量下降最为明显(32.6%±2.3%,P＜0.05),4 h细胞中IκBα含量较2 h有所上升,但仍然明显低于ArgⅡ刺激0 h细胞中IκBα的含量(40.1%±4.7%,P＜0.05).氯沙坦组NF-κB活性(115.4±10.7)和IκBα含量(43.6%±3.7%)与AngⅡ组0 h相比无明显差异.氯沙坦组中NF-κB活性明显低于AngⅡ组2 h,氯沙坦组中iκBα的含量亦明显高于AngⅡ组2 h.结论 AugⅡ能通过AT1介导HPMEC中NF-κB活化.经典途径参与了AagⅡ诱导的HPMEC中NF-κB活化过程.

Study of angiotensin-Ⅱactivating NF-κB in human pulmonary microvascular endothelial cells through classical pathway

Objective To investigate the activation of nuclear factor-κB(NF-κB),which was stimulated by angiotensin-Ⅱ(AngⅡ)through classical pathway in human pulmonary microvascular endothehal cells(HPMEC).Method The experiment was divided into two groups:in Ang Ⅱ group,HPMECS were incubated with 10-6mol/L AngⅡ for 0,0.5,1,2 and 4 hours,respectively;in losartan group,HPMEC was pretreated with 10-6mol/L losartan(inhibitor of AngⅡ type 1 receptor)for 1 hour,and then stimulated with 10-6mol/L AngⅡ for 2 hours,and the nucleax protein and the cell plasma protein were prepared by lysis and centrifugation.Electrophoretic mobility shift assay(EMSA)was used to detect the NF-κB DNA binding activity.The inhibitor of κBa(IκBα)was detected by Western blotting.The data were expressed as(x±s)and analyzed with one way analysis of variance.A P value less than 0.05 indicated significant difference.Results Compared with the activity of NF-κB at 0 h (100.0±25.1)after AngⅡstimalation,the activity increased significantly at 0.5 hour(144.5±16.1,P＜0.05),and reached peak value at 2 hours(270.1±27.2,P＜0.05).The concentration of IκBα at 0 hours was 44.4%±2.1%,decreased markedly at 0.5 hours(38.9%±3.6%,P＜0.05),and to the lowest level at 2hours(32.6%±2.3%,P＜0.05).The activity of NF-κB(115.4±10.7)and the concentration of IκBα(43.6%±3.7%)in losartan group had ilo significant difference with AngⅡ group at 0 h(P＞0.05).The activity of NF-κB and the concentration of IκBα in losartan group had significant difference with AngⅡ group at 2hours.Conclusions NF-κB can be activated through classical pathway,which stimulated by AngⅡ in HPMEC.