启膈散对人食管癌EC9706细胞增殖、凋亡及miR-133a/Akt/mTOR信号通路的影响  

Effect of Qigesan on Proliferation,Apoptosis and miR-133a/Akt/mTOR Signaling Pathway of Esophageal Cancer EC9706 Cells

作　　者：高小玲[1] 刘陆 李墨颜 李艳坤[1] 王蕊 陈玉龙[1] 崔姗姗[1] GAO Xiao-ling;LIU Lu;LI Mo-yan;LI Yan-kun;WANG Rui;CHEN Yu-long;CUI Shan-shan(Henan University of Chinese Medicine,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学,郑州450046

出　　处：《中国实验方剂学杂志》2021年第22期1-6,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81803981);河南省高等学校青年骨干教师培养项目(2018GGJS084)。

摘　　要：目的:观察启膈散对EC9706细胞增殖、凋亡的影响和对微RNA-133a(miR-133a)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(mTOR)信号通路的影响。方法:采用乙酸乙酯法提取启膈散有效部位;噻唑蓝(MTT)比色法筛选启膈散细胞用药剂量及检测启膈散对EC9706细胞增殖的影响;流式细胞仪检测启膈散对EC9706细胞凋亡的影响;实时荧光定量聚合酶链式反应(Real-time PCR)检测启膈散对miR-133a及胰岛素样生长因子1受体(IGF-1R)mRNA表达的影响;蛋白免疫印迹法(Western blot)检测启膈散对miR-133a/Akt/mTOR通路靶蛋白Akt,mTOR的表达。结果:与空白组比较,启膈散可抑制EC9706细胞的增殖(P<0.01),并呈剂量依赖性,筛选30%抑制浓度(IC30)40 mg·L^(-1)和半抑制浓度(IC50)80 mg·L^(-1)进行后续实验;与空白组比较,抑制剂组、抑制剂+启膈散组均可抑制EC9706细胞增殖(P<0.01),筛选抑制剂0.25μmol·L^(-1)进行后续实验;与空白组比较,启膈散80 mg·L^(-1)组可明显促进EC9706细胞晚期凋亡率及总凋亡率(P<0.05),启膈散40 mg·L^(-1)组可促进EC9706细胞晚期凋亡率(P<0.05),与Akt/mTOR抑制剂联合用药后,可协同增效。与空白组比较,各组均能提高miR-133a表达(P<0.05),其中抑制剂与中药抑制剂+启膈散组促进作用明显。与空白组比较,各组均能够均可明显降低Akt,mTOR蛋白表达(P<0.05),与单独用药比较,抑制剂与中药联合应用组Akt,mTOR的蛋白表达有所降低。结论:启膈散能够抑制食管癌EC9706细胞的生长,促进EC9706细胞的凋亡,其抑制机制可能与调控miR-133a的表达,影响其下游Akt/mTOR信号通路有关。Objective:To observe the effect of Qigesan on the proliferation and apoptosis of the human esophageal cancer cell EC9706, and the effect on miR-133 a/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway. Method: The effective constituent of Qigesan was extracted by ethyl acetate. Thiazolyl blue tetrazolium bromide(MTT)colorimetric assay was used to determine the dosage of Qigesan on cells and to detect the effect of Qigesan on the proliferation of EC9706 cells. The effect of Qigesan on apoptosis of EC9706 cells was detected by flow cytometry. The effect of Qigesan on miR-133 a and insulin-like growth factor 1 receptor(IGF-1 R)mRNA expression was detected by Real-time quantitative polymerase chain reaction(Real-time PCR). The protein expression of Akt and mTOR in EC9706 cells was detected by Western blot. Result: Qigesan can inhibit the proliferation of EC9706 cells in a dose-dependent manner(P<0.01).Inhibitory concentrations 30% inhibition concentration(IC30)40 mg·L^(-1) and median inhibition concentration(IC50)80 mg·L^(-1) were selected for follow-up experiments. Compared with the blank group,both the inhibitor group and the combination drug group can inhibit the proliferation of EC9706 cells(P<0.01). The inhibitor at0.25 μmol·L^(-1) was selected for subsequent experiments. Compared with the blank group,Qigesan 80 mg·L^(-1) dose group could significantly promote the late apoptosis rate and total apoptosis rate of EC9706 cells(P<0.05),and the 40 mg·L^(-1) dose group could significantly promote the late apoptosis rate of EC9706 cells(P<0.05),which shows synergistic effect after concomitant use with Akt/mTOR inhibitor(P<0.05). Compared with the blank control group,each group can effectively increase expression of miR-133 a(P<0.05). The combination of inhibitor and traditional Chinese medicine(TCM)has obvious promotion effect. Compared with blank control group,the expressions of Akt and mTOR were significantly decreased in each group(P<0.05). Compared with single medication,the expression

关 键 词：启膈散 EC9706细胞 增殖 凋亡 微RNA-133a(miR-133a)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(mTOR)信号通路 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]