基于PI3K/AKT/mTOR信号通路研究双氢青蒿素对人肝癌SMMC-7721细胞自噬的影响  

Autophagy Effect of Dihydroartemisinin on Human Liver Cancer SMMC-7721 Cells Based on PI3K/AKT/mTOR Signaling Pathway

作　　者：朱海洋[1] 孙会卿[1] 张淑凤[1] 吴贺文[2] ZHU Haiyang;SUN Huiqing;ZHANG Shufeng;WU Hewen(The Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou Henan China 450052;Henan Provincial People′s Hospital,Zhengzhou Henan China 450003)

机构地区：[1]郑州大学第五附属医院,河南郑州450052 [2]河南省人民医院,河南郑州450003

出　　处：《中医学报》2022年第4期830-836,共7页Acta Chinese Medicine

基　　金：河南省科技研发专项项目(172102310098)。

摘　　要：目的:研究双氢青蒿素(dihydroartemisinin, DHA)对人肝癌SMMC-7721细胞自噬的影响,并初步探讨其作用机制。方法:采用不同浓度(0μmol·L^(-1)、6.25μmol·L^(-1)、12.5μmol·L^(-1)、25μmol·L^(-1)、50μmol·L^(-1)、100μmol·L^(-1))DHA分别作用于SMMC-7721细胞24 h、48 h、72 h后,MTT法检测DHA对细胞增殖的影响,计算DHA对SMMC-7721细胞的半抑制浓度(inhibitory concentration 50,IC50),并于显微镜下观察细胞形态变化;SMMC-7721细胞随机分为对照组、DHA组、抑制剂组、激动剂组,DHA组以IC50剂量的DHA干预细胞,抑制剂组采用5 mmol·L^(-1)3-甲基腺嘌呤(3-methyladenine, 3-MA)与IC50剂量的DHA干预细胞,激动剂组采用100μg·L^(-1)激动剂胰岛素样生长因子-1(insulin-like growth factors-1,IGF-1)与IC50剂量的DHA干预细胞,对照组采用常规培养基干预细胞,培养48 h后,采用透射电镜及MDC染色观察细胞自噬情况;Western Blot检测细胞中磷脂酰肌醇3-激酶(phosphatidylinositide 3-kinase, PI3K)/蛋白激酶B(protein kinase B,AKT)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信号通路相关蛋白及自噬相关蛋白的表达水平。结果:不同浓度DHA干预SMMC-7721细胞24 h、48 h、72 h后,细胞增殖抑制率显著升高(P<0.05),细胞形态发生改变,且呈剂量和时间依赖性;与对照组比较,DHA组和抑制剂组可诱导SMMC-7721细胞自噬,PI3K、p-AKT和p-mTOR蛋白相对表达量显著降低(P<0.05),LC3-Ⅱ/LC3-I和Beclin1蛋白相对表达量显著升高(P<0.05);与DHA组、抑制剂组比较,激动剂组SMMC-7721细胞自噬减弱,PI3K、p-AKT和p-mTOR蛋白相对表达量显著升高(P<0.05),LC3-Ⅱ/LC3-I和Beclin1蛋白相对表达量显著降低(P<0.05)。结论:DHA可抑制人肝癌SMMC-7721细胞增殖,促进SMMC-7721细胞自噬,其作用机制可能与抑制PI3K/AKT/mTOR信号通路有关。Objective: To observe the effect of dihydroartemisinin(DHA) on autophagy of human liver cancer SMMC-7721 cells, and preliminarily to explore its mechanism of action.Methods: Different concentrations(0 μmol·L^(-1),6.25 μmol·L^(-1),12.5 μmol·L^(-1),25 μmol·L^(-1),50 μmol·L^(-1),100 μmol·L^(-1)) DHA were used to act on SMMC-7721 cells for 24 h, 48 h, 72 h, MTT method was used to detect the inhibitory effect of DHA on cells, the median lethal concentration(IC50) of DHA was calculated, and the changes in cell morphology under a microscope was observed.SMMC-7721 cells were randomly divided into control group, DHA group, The inhibitor group was treated with 5 mmol·L^(-1)3-methyladenine(3-methyladenine, 3-MA) and DHA at IC50 dose to intervene cells, and the agonist group was treated with 100 μg·L^(-1) agonist insulin-like growth factor 1(insulin-like growth factor 1).-like growth factors-1,IGF-1) and IC50 dose of DHA intervened cells, the control group was treated with conventional medium to intervene cells.And cells were cultured for 48 hours, and then autophagy was observed by transmission electron microscope and MDC staining.Western Blot was used to detect phosphatidylinositide 3-kinase(PI3 K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR) signaling pathway and autophagy protein expression levels.Results: After culturing for 24 h, 48 h, and 72 h, the proliferation inhibition rates of SMMC-7721 cells were significantly increased(P<0.05),and the cell morphology changed in a dose-and time-dependent manner.Compared with the control group, the DHA group and inhibitor group could induce autophagy in SMMC-7721 cells, and the relative expressions of PI3 K,p-AKT,and p-mTOR proteins were significantly decreased(P<0.05),The relative expression of LC3-II/LC3-I and Beclin1 proteins was significantly increased(P<0.05);Compared with the DHA group and the inhibitor group, the autophagy of SMMC-7721 cells in the agonist group was attenuated, and the relative expressions of PI3 K,p-AKT and p-mTOR proteins were

关 键 词：肝癌 双氢青蒿素 细胞自噬 PI3K/AKT/mTOR信号通路 

分 类 号：R285.5[医药卫生—中药学]