AIM: To evaluate the effects of angiotensin II ( Ang II ) on autophagy induction in vascular endo-thelial cells. METHODS: Human vascular endothelial EA. hy926 cells were used in the study. Intracellular reactive oxygen species ( ROS ) levels were detected by a microplate reader after the cells were treated with Ang II ( 10 - mol/L ) or Ang II combined with antioxidant N — acetyl — L — cysteine ( NAC, 50 μmol/L ) for 24 h. The protein levels of LC3 — II was detected by Western blotting after the cells were stimulated by different concentrations ( 10-8, 10-7 , 10-6 mol/L) of Ang II for 24 h or by Ang II ( 10 -7 mol/L ) for different time (Oh, 6 h, 12 h, 24 h, 36 h ). The number of autophago-somes was evaluated by fluorescence microscopy after stained with acridine orange. Similarly, the protein level of LC3 - II and the number of autophagosomes were detected after treated with Ang II( 10-7mol/L), Ang II combined with autophagy inhibitor 3 - methyladenine ( 3 - MA ) at concentration of 2 mmol/L or Ang II combined with NAC at concentration of 50 μmol/L. RESULTS: Intracellular ROS level and LC3 - II protein level were significantly increased ( P < 0. 05 ) after the cells were treated with Ang II , accompanied by the significant increase in the number of autophagosomes. Ang II — induced autophagy ( as showed both in LC3 — II protein level and autophagosomes ) was dramatically down — regulated by the treatment with 3 - MA or NAC in EA. hy926 cells ( P <0. 05 ). CONCLUSION: Ang I induces autophagy through elevating ROS levels in EA. hy926 cells.%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,50 μmol/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(50 μmol/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.
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