ERK1/2在Ang Ⅱ诱导的血管内皮细胞凋亡中的表达及意义

Effect of ERK1/2 Signal Transduction Pathway on Vascular Endothelial Cell Apoptosis Induced by AngiotensinⅡ

目的观察细胞外信号调节激酶1/2(ERK1/2)在血管紧张素Ⅱ(AngⅡ)诱导的内皮细胞中不同时点的表达变化,阐明血管内皮细胞凋亡对动脉粥样硬化诊治的意义。方法制备AngⅡRPMI1640培养液(1×10-6mol/L)培养人脐静脉内皮细胞,采用四甲基偶氮唑蓝比色法测定内皮细胞存活率,通过AnnexinV-FITC/PI双染流式细胞仪检测细胞凋亡率,Hochest33258荧光染色观察凋亡细胞形态学变化,利用细胞免疫化学法分析凋亡调控基因Bcl-2、Bax表达的变化,Western blot测定磷酸化ERK1/2水平。结果 AngⅡ诱导的内皮细胞凋亡率明显高于对照组(P<0.01);与对照组相比,Bcl-2mRNA表达呈持续性降低;Bcl-2/Bax比值下降,ERK1/2磷酸化水平于诱导12h时明显上升,18h达高峰(P<0.01),24h下降至稳定,总ERK1/2蛋白水平无明显变化。结论 ERK1/2信号转导途径参与AngⅡ诱导内皮细胞凋亡的发生、发展,并可能通过调控内皮细胞Bcl-2/Bax比值来实现。

Objective To explore the changes of extracellar signal-regulated protein kinase （ERK1/2） in endothelial cell apoptosis induced by angiotensinⅡ（Ang Ⅱ） at different time courses,and its possible molecular mechanism.Methods Human umbilical vein endothelial cells （HUVECs） were cultured in vitro and intervened by AngⅡ.HUVECs were divided into two groups,the control group and AngⅡ group （stimulated by AngⅡ10-6 mol/L for 24 h）.Flow cytometry with Annexin V-FITC/PI double staining and Hoechst33258 fluorescence staining were used to detect HUVECs apoptosis.The expressions of apoptosis-associated genes Bcl-2 and Bax were detected by immunocytochemistry and ERK1/2 levels were detected by Western blot at different time points.Results 10-6 mol/L Ang Ⅱ stimulation induced cell apoptosis.Expression of Bcl-2 mRNA decreased time-dependently,and the ratio of Bcl-2/Bax decreased markedly （P 0.05）.Phosphorylation of ERK1/2 began to increase and reached the peak at 18 h（P 0.01）.Conclusion Cell apoptosis may be an important factor of atherosclerosis.One of its molecular mechanisms might be associated with the decreased expression of Bcl-2 and the Bcl-2/Bax ratio.It’s probable that activated ERK1/2 signal pathway is involved in the pathological and physiological reaction in the apoptosis of endothelial cells induced by Ang Ⅱ.