橘红素通过调控AMPK/SIRT3信号轴影响胃癌AGS细胞自噬的机制  

Mechanism of tangeretin affecting autophagy in gastric cancer AGS cells by regulating AMPK/SIRT3 signaling axis

作　　者：李强[1] 王刚[1] 王凡[1] 李玉庆 LI Qiang;WANG Gang;WANG Fan;LI Yuqing(General Surgery Department of Tangshan Union Hospital,Tangshan 063000,China;Emergency Department of Tangshan Union Hospital,Tangshan 063000,China)

机构地区：[1]唐山市协和医院普外科,唐山063000 [2]唐山市协和医院急诊科,唐山063000

出　　处：《西北药学杂志》2022年第5期52-58,共7页Northwest Pharmaceutical Journal

基　　金：河北省2021年医学科学研究项目(编号:20210941)。

摘　　要：目的探讨橘红素(tangeretin,Tan)通过腺苷酸活化的蛋白激酶(AMP activated proteinkinase,AMPK)/沉默信息调节因子2相关酶3(silent information regulator factor 2related enzyme 3,SIRT3)信号轴对人胃癌AGS细胞自噬的影响及机制。方法用不同浓度(5、10、30、60、120μmol·L^(-1))Tan分别作用于AGS细胞24、48、72 h,用MTT法检测Tan对细胞的抑制作用,计算半抑制浓度(50% inhibitory concentration,IC_(50)),观察细胞形态变化情况;将细胞随机分为对照组、Tan组、激动剂组,其中Tan组、激动剂组分别用IC_(50)的Tan、AMPK激动剂A-769662处理细胞,对照组不做处理,培养48 h后,用透射电镜观察自噬泡,用MDC染色法观察细胞自噬情况,用qRT-PCR法检测AMPK以及SIRT3基因诱导量的变化,用Western blot检测细胞中相关蛋白AMPK、p-AMPK、SIRT3、LC3II、p62的表达水平。结果培养24、48、72 h后,AGS细胞对Tan的IC_(50)值分别为(46.42±5.62)、(59.76±7.01)、(83.56±8.86)μmol·L^(-1),不同浓度的Tan作用于细胞,细胞形态发生改变,随着Tan浓度的升高,细胞形态变化程度加剧;透射电镜下对照组有少量自噬泡形成,Tan组无自噬泡形成,激动剂组有大量自噬泡形成;MDC染色结果显示,与对照组比较,Tan组的荧光强度较弱,激动剂组的荧光强度较强;与对照组比较,Tan组SIRT3 mRNA的相对表达量降低,激动剂组SIRT3 mRNA的相对表达量升高(P<0.05)。与Tan组比较,激动剂组SIRT3 mRNA的相对表达量升高(P<0.05)。与对照组比较,Tan组细胞中p-AMPK、SIRT3、LC3II蛋白的相对表达量降低,p62蛋白的相对表达量升高,激动剂组p-AMPK、SIRT3、LC3II蛋白的相对表达量升高,p62蛋白的相对表达量降低(P<0.05)。与Tan组比较,激动剂组细胞中p-AMPK、SIRT3、LC3II蛋白的相对表达量升高,p62蛋白的相对表达量降低(P<0.05)。各组细胞中AMPK mRNA和蛋白的相对表达量比较,差异无统计学意义。结论Tan可抑制人胃癌AGS细胞发生自噬,可能�Objective To investigate the effect of tangeretin(Tan)on human gastric cancer AGS through the AMP-activated proteinkinase(AMPK)/silent information regulator factor 2 related enzyme 3(SIRT3)signaling axis,and the mechanism of autophagy.Methods Different concentrations(5,10,30,60,120μmol·L^(-1))of Tan were used to act on AGS cells for 24,48 and 72 hours.MTT method was used to detect Tan's inhibitory effect on cells,the half inhibitory concentration(IC_(50))of cells was calculated,and the cell morphology changes were observed.Tan intervention cells with IC_(50) concentration were randomly divided into control group,Tan group and agonist group.The Tan group and the agonist group were treated with Tan and AMPK agonist A-769662,respectively,while the control group were not treated.After 48 hours of culture,the autophagy vesicles were observed under transmission electron microscope,and the autophagy was observed by MDC staining.The changes of AMPK and SIRT3 gene induction were detected by using RT-qPCR method,and the expression of related proteins AMPK,p-AMPK,SIRT3,LC3II and p62 in cells were detected by using Western blot.Results After culturing for 24,48 and 72 hours,the IC_(50) values of AGS cells to Tan were(46.42±5.62),(59.76±7.01)and(83.56±8.86)μmol·L^(-1),respectively.Different concentrations of Tan acted on the cells,and the cell morphology changed.As the concentration of Tan increased,the degree of cell morphology changed much.Under transmission electron microscopy,a small number of autophagic vesicles formed in the control group,but no autophagic vesicles formed in the Tan group,and a large number of autophagic vesicles appeared in the agonist group.MDC staining showed that compared with the control group,the Tan group had weaker fluorescence intensity and the agonist group had stronger fluorescence intensity.Compared with the control group,the relative expression of SIRT3 mRNA in the Tan group decreased,and the relative expression of SIRT3 mRNA in the agonist group increased,and the difference was stat

关 键 词：橘红素 腺苷酸活化的蛋白激酶/沉默信息调节因子2相关酶3信号通路 胃癌 自噬 

分 类 号：R965[医药卫生—药理学]