南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (10): 1554-1561.doi: 10.12122/j.issn.1673-4254.2021.10.15

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白杨素通过抑制PI3K/AKT/mTOR信号通路发挥抗炎和抗氧化作用：基于蛋白质芯片方法

蔡苏娜，李 强，周 慧，许雨墨，宋 静，甘 超，戚之琳，齐世美   

  1. 皖南医学院生物化学与分子生物学教研室，活性大分子重点实验室，人体解剖学教研室，安徽 芜湖 241002
  • 出版日期:2021-10-20 发布日期:2021-11-11

Mechanism of PI3K/AKT/mTOR signaling pathway for mediating anti-inflammatory and anti-oxidant effects of chrysin: a protein microarray-based study

CAI Suna, LI Qiang, ZHOU Hui, XU Yumo, SONG Jing, GAN Chao, QI Zhilin, QI Shimei   

  1. Department of Biochemistry and Molecular Biology, Key Laboratory of Active Macromolecules, Department of Human Anatomy, Wannan Medical College, Wuhu 241002, China
  • Online:2021-10-20 Published:2021-11-11

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摘要： 目的 探讨PI3K/AKT/mTOR信号通路在白杨素抗炎抗氧化作用中的机制。方法 分别用0、5、10、15、30、60、120、240 μg/mL白杨素处理RAW264.7细胞24 h后，CCK-8法检测细胞活力。用白杨素预处理细胞2 h，加入脂多糖（100 ng/mL）分别刺激0、10、30 min，1、2、4、8、16 h，运用蛋白质芯片进行相关信号分子的筛选。用白杨素（10、30、60 μg/mL）孵育细胞2 h后，加入脂多糖刺激18 h，ELISA法检测IL-6，MCP-1和TNF-α的释放量；Griess法检测NO浓度；DCFH-DA荧光探针法检测ROS水平。设立空白对照组，白杨素（60 μg/mL）单独处理组，脂多糖（100 ng/mL）单独刺激组，以及白杨素和脂多糖联合处理组，RT-PCR法检测iNOS和COX-2的mRNA的表达量。分别用脂多糖（100 ng/mL），N-乙酰-半胱氨酸（NAC）（20 μmol/L）或白杨素（10、30、60 μg/mL）单独或共处理细胞后，用Western blot检测炎症相关通路p-AKT、p-PRAS40、p-mTOR、mTOR、p-P70S6k、p-S6RP、 S6RP的表达水平。结果 白杨素剂量在60 μg/mL内，对细胞活力基本无影响（P>0.05）；白杨素能够降低脂多糖刺激诱导的IL-6，MCP-1，TNF-α和炎症介质NO的释放量（P<0.01），抑制iNOS和COX-2的蛋白表达量和mRNA的表达水平（P<0.01）；蛋白质芯片筛选结果提示，脂多糖能够激活AKT/mTOR信号通路，而白杨素抑制其信号分子的活化，Western blot结果进一步验证了蛋白质芯片的结果（P<0.01）；白杨素显著下调内源性ROS的生成；运用NAC清除细胞内ROS后，炎症蛋白iNOS和COX-2的表达量下调（P<0.05），而AKT/mTOR通路的活化被阻断（P<0.05）。结论 白杨素通过抑制上游信号分子ROS的合成，进而抑制AKT/mTOR信号通路的活化，调控核糖体的翻译过程，下调促炎细胞因子和炎症介质的合成和释放，发挥抗炎作用。

关键词: 白杨素；蛋白质芯片；脂多糖；AKT/mTOR；ROS

Abstract: Objective To investigate the mechanism of PI3K/AKT/mTOR signaling pathway for mediating the anti-inflammatory and anti-oxidant effects of chrysin. Methods RAW264.7 cells were treated with different concentrations of chrysin for 24 h, and the changes in cell viability were detected using CCK-8 method. The cells with or without chrysin pretreatment for 2 h were stimulated with lipopolysaccharide (LPS) for different lengths of time, and the related signal molecules were screened using protein chip technique. In cells pretreated with chrysin for 2 h followed by LPS stimulation for 18 h, the release of IL-6, MCP-1 and TNF-α by the cells was detected with ELISA, and NO production was examined using Griess method, and ROS level was determined using DCFH-DA. The effects of chrysin, LPS, and their combination on the mRNA expressions of iNOS and COX-2 were detected using RT-PCR; Western blotting was performed to examine the changes in cellular expressions of p-AKT, p-PRAS40, p-mTOR, mTOR, p-P70S6k, p-S6RP and S6RP following the treatments with LPS, N-Acetyl-L-cysteine, and chrysin, alone or in combinations. Results Chrysin below 60 μg/mL did not significantly affect the viability of RAW264.7 cells (P>0.05). Chrysin treatment significantly reduced the release of IL-6, MCP-1, and TNF-α and the level of NO (P<0.01), and inhibited the mRNA and protein expressions of iNOS and COX-2 (P<0.01) in the cells. The results of protein chip screening suggested that LPS could activate the AKT/mTOR pathway, which was significantly inhibited by chrysin pretreatment, and the results were verified by Western blotting (P<0.01). Chrysin treatment significantly reduced the generation of endogenous ROS, and treatment with N-Acetyl-L-cysteine to eliminate intracellular ROS obviously reduced the expressions of iNOS and COX-2 (P< 0.05) and blocked the AKT/mTOR pathway (P<0.05). Conclusion Chrysin can inhibit the synthesis of the upstream signaling molecule ROS to inhibit the activation of AKT/mTOR signaling pathway, regulate the translation process of ribosomes, down-regulate the synthesis and release of pro-inflammatory cytokines and inflammatory mediators, and thus produce anti-inflammatory effects.

Key words: chrysin; protein chip; lipopolysaccharide; AKT/mTOR; ROS