基于AKT/mTOR通路探讨六君子汤抑制EC9706生长的作用机制  

Discussion on the mechanism of growth inhibition of Liujunzi Decoction on EC9706 based on AKT/mTOR pathway

作　　者：尚艺婉 武颖烁 孟丹华 陈星[1] 刘洋[1] 周哲旭 刘娅茹 胡啸博 陈玉龙[1,3] SHANG Yiwan;WU Yingshuo;MENG Danhua;CHEN Xing;LIU Yang;ZHOU Zhexu;LIU Yaru;HU Xiaobo;CHEN Yulong(Traditional Chinese Medicine School(Zhongjing School),Henan University of Chinese Medicine,Zhengzhou 450046,China;Institute of Basic Theory of Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China;Henan Key Laboratory of TCM Prescription and Syndrome in Signaling,Henan University of Chinese Medicine,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学中医学院(仲景学院),郑州450046 [2]中国中医科学院中医基础理论研究所,北京100700 [3]河南中医药大学河南省方证信号传导重点实验室,郑州450046

出　　处：《中华中医药杂志》2024年第1期180-185,共6页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.82074313)。

摘　　要：目的:探讨六君子汤对食管癌细胞EC9706的生长抑制作用机制。方法:噻唑蓝(MTT)法检测六君子汤对EC9706细胞的活性抑制作用,流式细胞术检测各组细胞凋亡和周期分布,Seahorse能量代谢分析系统检测细胞糖酵解速率和线粒体压力,实时荧光定量PCR(qRT-PCR)检测各组细胞哺乳动物雷帕霉素靶蛋白(mTOR)和蛋白激酶B1(AKT1)mRNA相对表达,Western Blot检测各组细胞磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、mTOR和AKT1/2/3蛋白相对表达。结果:六君子汤浓度为200、300、400μg/mL时可显著抑制EC9706细胞活性(P<0.01)。选定六君子汤150、200μg/mL用于后续实验。流式细胞术结果表明,六君子汤可显著促进EC9706细胞凋亡(P<0.01),六君子汤200μg/mL可显著延长细胞S期,缩短G2/M期(P<0.05);糖酵解速率检测结果显示,六君子汤200μg/mL可显著抑制EC9706细胞的基础糖酵解、补偿糖酵解和糖酵解潜能(P<0.05);线粒体压力测试结果显示,六君子汤150μg/mL可显著抑制EC9706细胞ATP合成耗氧、基础呼吸值和非线粒体耗氧(P<0.05,P<0.01),六君子汤200μg/mL可显著抑制EC9706细胞的ATP合成耗氧、线粒体最大耗氧能力、基础呼吸值、非线粒体耗氧和备用呼吸能力(P<0.01);qRT-PCR结果显示,六君子汤150、200μg/mL可使EC9706细胞mTOR mRNA相对表达显著降低(P<0.05,P<0.01);六君子汤200μg/mL使EC9706细胞AKT1mRNA相对表达显著降低(P<0.01)。Western Blot结果显示,六君子汤150、200μg/mL可显著抑制p-mTOR和AKT1/2/3蛋白相对表达(P<0.05,P<0.01),六君子汤200μg/mL可显著抑制mTOR蛋白相对表达(P<0.01)。结论:六君子汤可通过干预AKT/mTOR通路促进食管癌细胞EC9706凋亡,诱导细胞周期阻滞,抑制细胞的能量代谢和生长。Objective:To explore the mechanism of Liujunzi Decoction on the growth inhibition of esophageal carcinoma cell EC9706.Methods:MTT assay was used to detect the inhibitory effect of Liujunzi Decoction on EC9706 cells,flow cytometry was used to detect apoptosis and cycle distribution in each group,Seahorse energy metabolism analysis system was used to detect glycolysis rate and mitochondrial pressure of cells in each group,qRT-PCR was used to detect the relative expression of mammalian target of rapamycin(mTOR)and protein kinase B1(AKT1)mRNA in each group of cells,Western Blot was used to detect the relative expression of p-mTOR,mTOR and AKT1/2/3 protein in each group.Results:Liujunzi Decoction could inhibit the activity of EC9706 cells when the concentration was 200,300,400μg/mL(P<0.01);Liujunzi Decoction 150μg/mL and 200μg/mL were selected for follow-up experiments.Flow cytometry showed that Liujunzi Decoction could significantly promote the apoptosis of EC9706 cells(P<0.01).Liujunzi Decoction 200μg/mL could prolong the S phase and shorten the G2/M phase(P<0.05).The results of glycolysis rate test showed that Liujunzi Decoction 200μg/mL could inhibit basic glycolysis,compensate glycolysis and glycolysis potential of EC9706 cells(P<0.05).Mitochondrial stress test results showed that Liujunzi Decoction 150μg/mL inhibited ATP synthesis oxygen consumption,basal respiratory value and non-mitochondrial oxygen consumption in EC9706 cells(P<0.05,P<0.01).Liujunzi Decoction 200μg/mL inhibited ATP synthesis oxygen consumption,mitochondrial maximum oxygen consumption capacity,basic respiratory value,non-mitochondrial oxygen consumption and reserve respiratory capacity of EC9706 cells(P<0.01).qRT-PCR results showed that Liujunzi Decoction 150,200μg/mL significantly decreased the relative expression of mTOR mRNA in EC9706 cells(P<0.05,P<0.01),Liujunzi Decoction 200μg/mL significantly decreased the mRNA relative expression of AKT1 in EC9706 cells(P<0.01).Western Blot results showed that Liujunzi Decoction 50,200μg/mL s

关 键 词：食管癌 六君子汤 EC9706 蛋白激酶B 哺乳动物雷帕霉素靶蛋白 

分 类 号：R285[医药卫生—中药学]