血管紧张素(1-7)对血管紧张素Ⅱ诱导人脐静脉内皮细胞凋亡的影响及机制分析

Effects of angiotensin-(1-7) on the apoptosis of human umbilical vein endothelial cells induced by Ang Ⅱ

目的 观察血管紧张素（1-7）[Ang-（1-7）]对血管紧张素Ⅱ（Ang Ⅱ）诱导人脐静脉内皮细胞（HUVECs）凋亡的影响并探讨其可能的作用机制.方法 采用胰蛋白酶消化法原代培养HUVECs,取2～5代用于实验,培养的HUVECs随机分为：对照组、AngⅡ组、Ang-（1-7）组、AngⅡ＋Ang-（1-7）组、AngⅡ＋Ang-（1-7）＋A-779组,用吖啶橙（AO）/溴化乙啶（BE）法观察细胞凋亡的形态学变化,用流式细胞术检测内皮细胞的凋亡率 用Western blot方法检测p38丝裂原活化蛋白激酶（p38MAPK）磷酸化的表达水平.结果 （1）AngⅡ（10-6 mol/L）可以诱导HUVECs凋亡率增高,与对照组比较差异有统计学意义（25.60%±3.17%比2.32%±0.24%,P〈0.005） 不同浓度的Ang-（1-7）（10-9～10-6mol/L）呈剂量依赖性抑制AngⅡ所诱导的内皮细胞的凋亡,与AngⅡ组相比差异有统计学意义（均为P〈0.05） 加用Ang-（1-7）特异性受体拮抗剂A-779可阻断Ang-（1-7）的上述效应,与AngⅡ＋Ang-（1-7）组比较差异有统计学意义（23.37%±0.75%比7.79%±1.50%,P〈0.05） （2）与对照组相比,AngⅡ（10-6 mol/L）诱导后HUVECs p38MAPK磷酸化表达水平增加（P〈0.05） 不同浓度的Ang-（1-7）（10-9～10-6mol/L）呈剂量依赖性抑制AngⅡ所诱导的p38MAPK磷酸化表达水平,与AngⅡ组相比,差异有统计学意义（均为P〈0.05）,加用A-779可阻断Ang-（1-7）的上述效应,与AngⅡ＋Ang-（1-7）组比较差异有统计学意义（P〈0.05）.结论 AngⅡ可诱导内皮细胞凋亡及p38MAPK磷酸化表达增高,Ang-（1-7）呈浓度依赖性抑制AngⅡ的上述效应,并且是通过其特异性受体Mas发挥作用.

Objective To investigate the effect of angiotensin（Ang）-（ 1-7 ） on apoptosis of Human umbilical vein endothelial ceils （HUVECs） induced by Ang II. Methods HUVECs were isolated and cultured. Cultured human HUVECs were incubated for 24 h with Ang-（ 1-7）, Aug U, Ang-（1-7） ＋Ang II and A-779 ＋Ang lI ＋Ang-（1-7） respectively. Cultured HUVECs without incubating stimulator were chosen as controls. The apoptosis of endothelial cells were detected by flow cytometry. The expression of the pbosphorylation of p38MAPK was determined by Western blot. Results （ 1 ） The apoptosis rate of HUVECs was significantly higher in Aug II group than in control group （ 25. 60% ± 3. 17% vs. 2. 32% ± 0. 24% ,P 〈 0. 005）. Compared with Aug Ⅱ group, Ang-（ 1-7 ） inhibited the apoptosis of HUVECs in a dose dependent manner （ P 〈 0. 05 ）. This effect was abolished by A-779, a specific receptor antagonist of Ang-（ 1-7 ）. The apoptosis rate of HUVECs was significantly higher than Ang Ⅱ ＋Ang-（1-7） group （23.37% ±0. 75% vs. 7. 79% ± 1.50% ,P 〈0. 05）. （2） Compared with control group, the expression of the phosphorylation of p38MAPK was significantly increased in Angllgroup （P 〈0. 05）. And significantly inhibited by Ang-（1-7） group in a dose depent manner. The expression of the phosphorylation of p38 MAPK was reduced in Ang Ⅱ ＋Ang- （ 1-7 ） group compared with Ang lI group （ P 〈 0. 05 ）. The effect of Ang-（ 1-7 ） was weakened when adding A-779 into Ang II ＋ Aug- （ 1-7 ） cultured HUVECs. The level of the phosphorylation of p38 MAPK was significantly higher in Aug Ⅱ ＋Ang- （ 1-7 ） ＋ A-779 group than in Ang Ⅱ ＋Ang- （ 1-7 ） group （P 〈 0.05）. Conclusions Ang Ⅱ induces apoptosis of HUVECs in vitro and increases the expression of the phosphorylation of p38MAPK. Ang-（1-7） attenuates above effects of Ang Ⅱ in a dose dependent manner and probably via its specific receptor Mas.