蛇葡萄素通过PI3K/AKT/mTOR通路诱导人宫颈癌细胞SiHa自噬  

Ampelopsin induces autophagy in human cervical cancer SiHa cells through PI3K/AKT/mTOR pathway

作　　者：邹雪 熊晓妹 杨晓利 廖思雨 许诗怡 张秀桥 桂春 ZOU Xue;XIONG Xiao-mei;YANG Xiao-li;LIAO Si-yu;XU Shi-yi;ZHANG Xiu-qiao;GUI Chun(College of Pharmacy,Hubei University of Chinese Medicine,Wuhan 430065,China)

机构地区：[1]湖北中医药大学药学院,湖北武汉430065

出　　处：《中国药理学通报》2023年第11期2121-2128,共8页Chinese Pharmacological Bulletin

基　　金：湖北省科技重大专项(No 2020ACA007);武汉市科技局应用基础前沿项目(No 2018060401011308)。

摘　　要：目的研究蛇葡萄素(ampelopsin,AMP)诱导人宫颈癌细胞SiHa发生自噬及其可能的作用机制。方法单丹磺酰胺(monodansylcadaverine,MDC)染色法观察AMP对SiHa和C-33A细胞自噬的影响;透射电子显微镜(transmission electron microscope,TEM)观察经AMP干预的SiHa和C-33A细胞超微结构的变化;自噬双标腺病毒转染(mRFP-GFP-LC3)法检测SiHa细胞自噬流强度;Western blot法对SiHa细胞中自噬相关蛋白LC3Ⅱ/Ⅰ、P62、Beclin-1、Atg13及PI3K/AKT/mTOR信号通路蛋白的表达情况进行检测。结果MDC染色结果显示,随着AMP浓度的升高,代表自噬小体的亮绿色逐渐增多;TEM实验结果表明,与空白对照组相比,AMP组细胞线粒体嵴溶解消失,同时可见自噬小体和自噬溶酶体;mRFP-GFP-LC3实验结果提示,随着AMP浓度的升高,细胞内自噬小体和自噬溶酶体增多,提示自噬流增强;同时发现LC3Ⅱ/Ⅰ、Beclin-1、Atg13蛋白表达上调,P62、p-PI3K、p-AKT、p-mTOR蛋白表达下调,表明AMP可能通过抑制PI3K/AKT/mTOR信号通路诱导SiHa细胞发生自噬;加入3-MA后,与AMP组相比,3-MA和AMP联合组的p-PI3K、p-AKT、p-mTOR的表达水平上升,提示自噬被抑制后,PI3K/AKT/mTOR信号通路被逆转。结论AMP可诱导SiHa细胞发生自噬,其机制可能与抑制PI3K/AKT/mTOR信号通路的激活有关。Aim To explore the autophagy of human cervical cancer SiHa cells induced by ampelopsin(AMP)and its possible mechanism.Methods The monodansylcadaverine(MDC)method was used to observe the effect of AMP on autophagy of SiHa and C-33A cells.Transmission electron microscope(TEM)was employed to observe the ultrastructure of SiHa and C-33A cells after AMP intervention.mRFP-GFP-LC3 transfection was applied to detect autophagy flux in SiHa cells.Western blot was used to detect the expressions of autophagy related proteins LC3Ⅱ/Ⅰ,P62,Beclin-1,Atg13 and PI3K/AKT/mTOR signaling pathway proteins.Results The number of bright green autophagosome indicated gradual increase with the increase of AMP concentration.The results of TEM showed that compared with the blank control group,the mitochondrial cristeolysis disappeared,and autophagosomes and autophagolysosomes were observed in AMP group.The mRFP-GFP-LC3 transfection experiment indicated that the intracellular autophagosomes and autophagolysosomes increased with the increase of AMP concentration,and the autophagy flux was enhanced.AMP induced autophagy by up-regulating LC3Ⅱ/Ⅰ,Beclin-1 and Atg13,and down-regulating the expression of P62,p-PI3K,p-AKT and p-mTOR.Furthermore,the expression levels of p-PI3K,p-AKT and p-mTOR increased after the addition of autophagy inhibitor 3-MA.Conclusions AMP can induce autophagy in SiHa cells,which may be related to inhibiting the activation of PI3K/AKT/mTOR signaling pathway.

关 键 词：蛇葡萄素 宫颈癌 SIHA细胞 C-33A细胞 自噬 PI3K/AKT/MTOR 

分 类 号：R282.71[医药卫生—中药学] R392[医药卫生—中医学] R394.2R737.3