Objective
To investigate the mechanism by which angiotensin Ⅱ-induced oxidative stress response inhibits AMPK/ SIRT1 signaling in RAW264.7 macrophages. Objective: RAW264.7 cells were treated with 0.5, 1, 3, 10, or 20 μmol/L angiotensin Ⅱ for 24 h, and the changes in the expressions of AMPK, p-AMPK, and SIRT1 proteins were detected using Western blotting. The intracellular ROS release level was measured and the levels of SOD and MDA were detected. The effects of angiotensin Ⅱ type 1 receptor (AT1R) gene silencing on the cell response to angiotensin Ⅱ treatment were examined by detecting the changes in AMPK, p-AMPK and SIRT1 protein levels. The effects of a ROS inhibitor on cellular AMPK and SIRT1 were also examined. Objective: Angiotensin Ⅱ stimulation at 20 μmol/L significantly inhibited the phosphorylation of AMPK protein and increased cellular ROS release (P < 0.05). Treatment with 0.5-10 μmol/L angiotensin Ⅱ did not cause significant changes in SOD activity or MDA expression, but angiotensin Ⅱ at the dose of 20 μmol/L significantly inhibited SOD activity in the cells (P < 0.05). In the macrophages with AT1R gene silencing, treatment with angiotensin Ⅱ did not obviously inhibit AMPK phosphorylation or down- regulate SIRT1 expression. In cells treated with the ROS inhibitor, angiotensin Ⅱ failed to lower the level of AMPK phosphorylation or the expression of SIRT1. Objective: Angiotensin Ⅱ induces oxidative stress to cause disturbance of AMPK/ SIRT1 signaling pathway in macrophages. 目的: 探讨Angiotensin Ⅱ诱导巨噬细胞氧化应激反应与AMPK/SIRT1通路活化关系的机制。 方法: RAW264.7细胞正常培养后给予不同浓度的Angiotensin Ⅱ（0、0.5、1、3、10、20 μmol/L）处理24 h后，Western blot检测AMPK，p-AMPK和SIRT1的表达水平变化，和用DCFH探针检测ROS水平的变化，试剂盒检测细胞上清液中SOD活性和MDA表达量；同时采用基因编辑技术将Angiotensin Ⅱ的受体AT1R成功沉默后给予Angiotensin Ⅱ刺激，检测对AMPK，p-AMPK和SIRT1蛋白水平的影响以及使用ROS的抑制剂来观察细胞AMPK和SIRT1的变化情况。 结果: 20 μmol/L的Angiotensin Ⅱ的刺激能显著抑制蛋白AMPK的磷酸化（P < 0.05），抑制SIRT1的表达；同时增加了细胞ROS的释放（P < 0.05）。在检测SOD活性和MDA表达量时，0.5~10 μmol/L的Angiotensin Ⅱ对细胞无明显改变（P>0.05），20 μmol/L的Angiotensin Ⅱ明显抑制SOD活性（P < 0.05），能显著增加MDA的产生。沉默了AT1R后，Angiotensin Ⅱ不能抑制AMPK蛋白磷酸化以及对SIRT1的表达无明显下调作用；使用ROS抑制剂后，Angiotensin Ⅱ处理无法降低细胞磷酸化AMPK和SIRT1的表达。 结论: Angiotensin Ⅱ通过诱导巨噬细胞发生氧化应激反应从而引起AMPK/SIRT1信号通路的紊乱。