血管紧张素Ⅱ升高血管内皮细胞中ROS水平并激活自噬通路  

AngiotensinⅡ activates autophagy pathway through elevating ROS level in vascular endothelial cells

作　　者：范俊[1] 杨成明[1] 连继勤[2] 曾春雨[1] 倪振洪[2] 林德胜[1] 冉希[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所心血管内科,重庆400042 [2]第三军医大学基础部生物化学与分子生物学教研室,重庆400038

出　　处：《中国病理生理杂志》2012年第7期1166-1171,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30371363)

摘　　要：目的:研究血管紧张素Ⅱ(AngⅡ)对血管内皮细胞自噬的影响及其可能机制。方法:以EA.hy926血管内皮细胞为研究对象,荧光酶标仪检测AngⅡ(10-7mol/L)、AngⅡ联合N-乙酰半胱氨酸(NAC,50μmol/L)作用24 h后细胞中活性氧簇(ROS)水平;用不同浓度(10-8、10-7、10-6mol/L)AngⅡ作用24 h或10-7mol/L AngⅡ作用不同时间(0 h、6 h、12 h、24 h、36 h)后,通过Western blotting分析微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)蛋白变化、吖啶橙染色结合荧光显微镜观察自噬体形成情况来判断细胞中自噬发生情况;AngⅡ(10-7mol/L)联合自噬抑制剂3-甲基腺嘌呤(3-MA,2 mmol/L)或NAC(50μmol/L)处理24 h后用同样方法检测细胞中自噬的发生情况。结果:细胞经AngⅡ刺激后细胞内ROS的水平显著升高(P<0.05),LC3-Ⅱ蛋白表达明显增强(P<0.05),自噬体形成明显增加;3-MA或NAC可显著抑制内皮细胞中AngⅡ诱导的LC3-Ⅱ蛋白表达(P<0.05)与自噬体形成。结论:AngⅡ可以通过升高血管内皮细胞内ROS的水平从而诱导自噬的发生。AIM： To evaluate the effects of angiotensin Ⅱ （ Ant Ⅱ） on autophagy induction in vascular endo- thelial cells. METHODS： Human vascular endothelial EA. hy926 cells were used in the study. Intracellular reactive oxy- gen species （ROS） levels were detected by a microplate reader after the cells were treated with Ant Ⅱ （ 10 ^-7 mol/L） or Ant Ⅱ combined with antioxidant N - acetyl - L - cysteine （ NAC, 50 μmol/L） for 24 h. The protein levels of LC3 - I1 was detected by Western blotting after the cells were stimulated by different concentrations （ 10 -8, 10 -7, 10 -6 mol/L） of AnglI for24 h orby Angll （10-Tmol/L） for different time （0 h, 6 h, 12 h, 24 h, 36 h）. The number of antophagnsomes was evaluated by fluorescence microscopy after stained with acridine orange. Similarly, the protein level of LC3 - Ⅱ and the number of autophagnsomes were detected after treated with Ant Ⅱ （10^-7mol/L）, Ant Ⅱ combined with autophagy inhibitor 3 - methyladenine （ 3 - MA） at concentration of 2 mmoL/L or Ant Ⅱ combined with NAC at concentration of 50 p, mol/L. RESULTS： Intracellular ROS level and LC3 - Ⅱ protein level were significantly increased （P 〈 0.05 ） after the cells were treated with Ant Ⅱ , accompanied by the significant increase in the number of autophagosomes. Ant Ⅱ - induced autophagy （ as showed beth in LC3 - Ⅱ protein level and autophagnsomes） was dramatically down - regulated by the treat- ment with 3 - MA or NAC in EA. hy926 cells （P 〈 0.05）. CONCLUSION： Ant Ⅱ induces autophagy through elevating ROS levels in EA. hy926 cells.

关 键 词：自噬 血管紧张素Ⅱ 血管内皮细胞 活性氧簇 氧化性应激 

分 类 号：R363[医药卫生—病理学]