南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (3): 384-390.doi: 10.12122/j.issn.1673-4254.2021.03.10

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Angiotensin II通过氧化应激引起巨噬细胞AMPK/SIRT1能量信号紊乱

肖 珊，马郁文，李 婧，张彦红，何 泓，方春香，王万铭   

  • 出版日期:2021-03-20 发布日期:2021-04-06

Angiotensin II inhibits AMPK/SIRT1 pathway by inducing oxidative stress in RAW264.7 macrophages

  • Online:2021-03-20 Published:2021-04-06

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摘要： 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。

关键词: 血管紧张素2；AMPK/SIRT1；RAW264.7；氧化应激

Abstract: Objective To investigate the mechanism by which angiotensin II-induced oxidative stress response inhibits AMPK/SIRT1 signaling in RAW264.7 macrophages. Methods 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. Results 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. Conclusion Angiotensin II induces oxidative stress to cause disturbance of AMPK/SIRT1 signaling pathway in macrophages.

Key words: angiotensin II; AMPK/SIRT1; RAW264.7 macrophages; oxidative stress