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、240μg/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、60μg/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(60μg/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)(20μmol/L)或白杨素(10、30、60μg/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60μg/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。
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-αby 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 60μg/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-αand 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.
作者
蔡苏娜
李强
周慧
许雨墨
宋静
甘超
戚之琳
齐世美
CAI Suna;LI Qiang;ZHOU Hui;XU Yumo;SONG Jing;GAN Chao;QI Zhilin;QI Shimei(Department of Biochemistry and Molecular Biology,Wannan Medical College,Wuhu 241002,China;Key Laboratory of Active Macromolecules,Wannan Medical College,Wuhu 241002,China;Department of Human Anatomy,Wannan Medical College,Wuhu 241002,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2021年第10期1554-1561,共8页
Journal of Southern Medical University
基金
安徽省大学生创新创业训练计划(201910368011,201910368022)
安徽高校自然科学研究项目重大项目(KJ2019ZD31,KJ2020ZD54)
皖南医学院重点科研项目培育基金(WK2018Z09)
活性生物大分子研究安徽省重点实验室项目(1306C083008)。
