N-乙酰-L-半胱氨酸通过AMPK/mTOR途径激活保护性自噬降低青蒿琥酯诱导的胰腺癌细胞死亡  

N-acetylcysteine reduces artesunate-induced pancreatic carcinoma cell death by activating protective autophagy via the AMPK/mTOR pathway

作　　者：任自敬 徐红霞 李星阅 王越[2] 马佳佳 周佩洋 REN Zijing;XU Hongxia;LI Xingyue;WANG Yue;MA Jiajia;ZHOU Peiyang(Xiangyang First People’s Hospital Affiliated to Hubei University of Medicine,Xiangyang 441000;Hubei University of Medicine,Shiyan 442000,China)

机构地区：[1]湖北医药学院附属襄阳市第一人民医院,湖北襄阳441000 [2]湖北医药学院,湖北十堰442000

出　　处：《西安交通大学学报（医学版）》2022年第3期354-360,共7页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金青年项目(No.81903005);襄阳市科技局项目(2020YL20);襄阳市第一人民医院科技创新项目。

摘　　要：目的使用活性氧(ROS)清除剂N-乙酰-L-半胱氨酸(NAC)探究青蒿琥酯(ART)对胰腺癌(PC)的抑制作用及机制。方法ART干预3种PC细胞系CFPAC-1、Capan-2及BxPC3,通过CCK8法检测细胞活性;Transwell法检测细胞迁移能力;Western blotting检测迁移相关蛋白E钙黏蛋白(E-cadherin)、N钙黏蛋白(N-cadherin)及波形蛋白(Vimentin)的表达;ROS探针DCFH-DA检测胞内ROS;用LC3细胞免疫荧光检测胞内自噬小体的形成;加入NAC或自噬抑制剂3-MA后,再通过CCK8法检测细胞活性,Western blotting检测p-AMPK/AMPK、p-mTOR/mTOR、p62及LC3Ⅱ/Ⅰ的表达。结果ART以时间、剂量依赖的方式抑制CFPAC-1、Capan-2的生长。200μmol/L ART处理CFPAC-1、Capan-2细胞48 h后,E-cadherin表达显著上调,N-cadherin及Vimentin显著下调,细胞迁移能力降低;ART上调胞内ROS水平,促进细胞自噬小体形成;NAC可逆转ART对CFPAC-1、Capan-2细胞的抑制效应,并上调p-AMPK/AMPK、p62、LC3Ⅱ/Ⅰ,下调p-mTOR/mTOR的表达,增强细胞自噬水平。3-MA不能逆转ART对PC细胞的抑制效应。结论ART依赖ROS,而非自噬发挥抗胰腺癌效应。NAC通过AMPK/mTOR信号通路激活保护性自噬,削弱ART对PC细胞的抑制。Objective In this study,reactive oxygen species(ROS)scavenger N-acetyl-L-cysteine(NAC)was used to explore the inhibitory effect and mechanism of artesunate(ART)on pancreatic carcinoma(PC)cells.Methods Different concentrations of ART interfered with 3 PC cell lines CFPAC-1,Capan-2 and BxPC3.Cell viability was measured by CCK8;cell migration ability was measured by Transwell method,and the expressions of migration-related proteins E-cadherin,N-cadherin and Vimentin were measured by Western blotting.ROS probe DCFH-DA was used to measure intracellular ROS;LC3 cell immunofluorescence(IF)was used to detect the formation of intracellular autophagosomes.After adding NAC or autophagy inhibitor 3-MA,the cell viability was tested again by CCK8,and the expressions of p-AMPK/AMPK,p-mTOR/mTOR,p62 and LC3Ⅱ/Ⅰwere detected by Western blotting.Results ART inhibited the growth of CFPAC-1 and Capan-2 in a time-and dose-dependent manner.After treatment of CFPAC-1 and Capan-2 cells with 200μmol/L of ART for 48 h,the expression of E-cadherin was upregulated,while N-cadherin and Vimentin were downregulated,and the cell migration ability was significantly reduced.ART significantly upregulated intracellular ROS level and promoted the formation of autophagosomes.NAC could reduce the inhibitory effect of ART on CFPAC-1 and Capan-2 cells,upregulate p-AMPK/AMPK,P62 and LC3Ⅱ/Ⅰ,downregulate the expression of p-mTOR/mTOR,and intensify autophagy.3-MA could not reverse the inhibitory effect of ART on PC cells.Conclusion ART is dependent on ROS,but not on autophagy,in exerting an anti-pancreatic carcinoma effect.NAC attenuates the inhibitory effect of ART on PC cells by activating protective autophagy through AMPK/mTOR signaling pathway.

关 键 词：青蒿琥酯 胰腺癌 活性氧 自噬 AMPK/mTOR 

分 类 号：R735.9[医药卫生—肿瘤]