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

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。

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

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.