刺五加苷B介导PI3K/Akt/mTOR通路诱导肺癌细胞凋亡和自噬  

Eleutheroside B induces apoptosis and autophagy of lung cancer cells by regulating PI3K/Akt/mTOR pathway

作　　者：曹雪婷 吴博雅 陈静 CAO Xue-ting;WU Bo-ya;CHEN Jing(Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases,Hebei Key Laboratory for Chronic Diseases,School of Basic Medical Sciences,North China University of Science and Technology,Tangshan 063210,China;College of Life Sciences,North China University of Science and Technology,Tangshan 063210,China)

机构地区：[1]华北理工大学基础医学院,河北省慢性疾病基础医学重点实验室,唐山市慢性病临床基础研究重点实验室,河北唐山063210 [2]华北理工大学生命科学学院,河北唐山063210

出　　处：《中国中药杂志》2023年第24期6693-6701,共9页China Journal of Chinese Materia Medica

基　　金：河北省自然科学基金项目(H2021209004)。

摘　　要：探讨刺五加苷B对肺癌A549和H460细胞凋亡和自噬的影响及其分子机制。采用噻唑蓝比色法(methyl thiazolyl tetrazolium colorimetric,MTT)检测5、10、15、20、25、30、35、40、45 mmol·L^(-1)刺五加苷B对肺癌细胞的细胞毒作用;台盼蓝排斥实验检测不同时间刺五加苷B对肺癌细胞A549和H460存活率的影响;克隆形成实验检测刺五加苷B对肺癌细胞A549和H460增殖的影响;acridine orange/ethidium bromide(AO/EB)荧光双染、Hoechst 33342荧光染色法检测刺五加苷B对肺癌细胞A549和H460凋亡的影响,并应用Western blot法检测凋亡相关蛋白探究凋亡相关分子机制;应用AO荧光染色、Western blot法检测自噬泡以及自噬相关蛋白P62和微管相关蛋白1轻链3蛋白(microtubule-associated protein 1 light chain 3,LC3)的表达情况。结果显示,与对照组相比,刺五加苷B以浓度依赖的方式抑制肺癌细胞A549和H460的生长,并得到刺五加苷B对肺癌细胞A549和H460最佳作用时间均为24 h,最佳作用浓度分别为28.64、22.16 mmol·L^(-1);刺五加苷B能够抑制肺癌细胞A549和H460集落形成;与对照组相比,在刺五加苷B的作用下能够促进凋亡小体的形成诱导细胞发生凋亡,同时诱导线粒体途径相关蛋白的表达;在刺五加苷B的作用下肺癌细胞中酸性自噬泡增多,LC3Ⅱ表达增加、P62蛋白表达下降,磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)/丝氨酸-苏氨酸激酶(serine-threonine kinase,Akt)/哺乳动物雷帕霉素靶标(mammalian target of rapamycin,mTOR)通路中PI3K、p-Akt和p-mTOR蛋白表达下降。研究表明,刺五加苷B能够抑制肺癌细胞生长,减少集落形成,并发现是通过线粒体途径诱导肺癌细胞发生凋亡,此外还可诱导细胞自噬的发生,其作用机制可能与PI3K/Akt/mTOR通路有关。This study investigated the effect of eleutheroside B on apoptosis and autophagy of lung cancer A549 and H460 cells and its molecular mechanism.MTT assay was used to detect the cytotoxicity of eleutheroside B at 5,10,15,20,25,30,35,40,and 45 mmol·L^(-1) on lung cancer cells.Trypan blue exclusion assay was used to detect the effect of eleutheroside B on the survival rate of lung cancer A549 and H460 cells at different time.Colony formation assay was used to detect the effect of eleutheroside B on the proliferation of lung cancer A549 and H460 cells.AO/EB fluorescence double staining and Hoechst 33342 fluorescence staining were used to detect the effect of eleutheroside B on apoptosis of lung cancer A549 and H460 cells,and Western blot was used to detect apoptosis-related proteins to explore the apoptosis-related molecular mechanism.AO fluorescence staining and Western blot were used to detect the expression of autophagic vesicles and autophagy-related proteins P62 and LC3.The results showed that compared with the control group,eleutheroside B inhibited the growth of lung cancer A549 and H460 cells in a concentration-dependent manner.The optimal effect time of eleutheroside B on lung cancer A549 and H460 cells was 24 h,and the optimal concentrations were 28.64 and 22.16 mmol·L^(-1),respectively.Eleutheroside B could inhibit the colony formation of A549 and H460 cells.Compared with the control group,eleutheroside B could promote the formation of apoptotic bodies and induce cell apoptosis,as well as induce the expression of mitochondrial pathway-related proteins.Under the effect of eleutheroside B,the acidic autophagy vacuole in lung cancer cells increased,LC3Ⅱexpression increased,P62 protein expression decreased,and PI3K,p-Akt,and p-mTOR protein expression decreased in the PI3K/Akt/mTOR pathway.Studies have shown that eleutheroside B can inhibit the growth of lung cancer cells,reduce colony formation,induce apoptosis of lung cancer cells through mitochondrial pathway,and induce autophagy.The mechanism may be rel

关 键 词：刺五加苷B 肺癌 PI3K/Akt/mTOR通路 凋亡 自噬 

分 类 号：R285[医药卫生—中药学]