基于自噬抑制剂3-MA介导的大叶蛇葡萄总黄酮提取物对人乳腺癌细胞增殖、凋亡的影响  

Effects of total flavone extract from Ampelopsis megalophylla mediated by autophagy inhibitor 3-MA on proliferation and apoptosis of human breast cancer cells

作　　者：许诗怡 廖思雨 张天旭 邹雪 桂春 张秀桥 XU Shi-yi;LIAO Si-yu;ZHANG Tian-xu;ZOU Xue;GUI Chun;ZHANG Xiu-qiao(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》2024年第6期1115-1123,共9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);湖北中医药大学青苗计划项目(No 2020ZZX028)。

摘　　要：目的探讨抑制自噬时,大叶蛇葡萄总黄酮提取物(total flavonoid extract,TFE)对人乳腺癌细胞增殖、凋亡的影响及其作用机制。方法针对人宫颈癌细胞Hela、人肺癌细胞A549、人肝癌细胞SMMC-7721、人乳腺癌细胞MCF-7、MDA-MB-231及人正常肝细胞L-02,采用MTT法优选敏感细胞株;通过TFE与自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)联合运用,采用MTT法检测其对敏感细胞增殖的抑制作用;透射电子显微镜及Hoechst 33258单染法观察细胞的形态学变化;Annexin V-FITC/PI双染法检测细胞凋亡率的变化;Western blot检测凋亡相关蛋白和通路蛋白(死亡受体途径、线粒体途径、内质网应激途径)的表达水平;免疫荧光法检测线粒体途径关键蛋白Cyt-c表达,并选择自噬激动剂雷帕霉素(rapamycin,RA)进行反向验证。结果TFE可浓度依赖性抑制人乳腺癌细胞增殖,MCF-7细胞为敏感细胞株,与TFE组相比,TFE+3-MA组在24、48、72 h对MCF-7细胞的抑制率均明显增加(P<0.01),细胞数量减少、间隙增大,凋亡小体增多,凋亡率升高(P<0.01),Bax/Bcl-2(P<0.01)、cleaved caspase-3(P<0.01)、Cyt-c(P<0.05)、FADD、cleaved caspase-12的表达量均升高,核内凋亡蛋白Cyt-c表达增加;TFE+RA组荧光减弱,逆转了TFE诱导的线粒体途径凋亡。结论TFE能明显抑制人乳腺癌细胞的增殖,抑制自噬时可能通过线粒体途径促进MCF-7细胞凋亡,激活自噬可逆转细胞凋亡。Aim To explore the effect of total flavonoid extract(TFE)of Ampelopsis megalophylla on the proliferation and apoptosis of human breast cancer cells and its mechanism in autophagy inhibition.Methods For human cervical cancer cell Hela,human lung cancer cell A549,human liver cancer cell SMMC-7721,human breast cancer cell MCF-7,MDA-MB-231 and human normal liver cell L-02,MTT method was used to select sensitive cell lines.The inhibitory effect of TFE combined with autophagy inhibitor 3-methyladenine(3-MA)on sensitive cell proliferation was detected using MTT assay.The morphological changes of cells were observed using transmission electron microscopy and Hoechst 33258 single staining method.The changes in cell apoptosis rate were detected using Annexin V-FITC/PI dual staining method.The expression levels of apoptosis related proteins and pathway proteins(death receptor pathway,mitochondrial pathway,endoplasmic reticulum stress pathway)were detected uisng Western blot.The expression of the key protein Cyt-c in mitochondrial pathway was determiend by immunofluorescence,and the autophagy agonist rapamycin was selected for reverse validation.Results TFE could inhibit the proliferation of human breast cancer cells in a concentration-dependent manner,and MCF-7 cells were sensitive cell lines.Compared with the TFE group,the TFE+3-MA group significantly increased the inhibition rate of MCF-7 cells at 24,48,and 72 h(P<0.01).The number of cells decreased,the gap increased,the number of apoptotic bodies increased,and the apoptosis rate increased(P<0.01).The expression levels of Bax/Bcl-2(P<0.01),cleaved-caspase3(P<0.01),Cyt-c(P<0.05),FADD,and cleaved-caspase-12 all increased,and the expression of apoptotic protein Cyt-c in nucleus increased.The fluorescence of the TFE+RA group decreased,reversing the mitochondrial pathway apoptosis induced by TFE.Conclusions TFE can significantly inhibit the proliferation of human breast cancer cells.When inhibiting autophagy,it may promote the apoptosis of MCF-7 cells through the mitochondrial

关 键 词：大叶蛇葡萄总黄酮提取物 抑制自噬 乳腺癌细胞 MCF-7细胞 增殖 凋亡 

分 类 号：R284.1[医药卫生—中药学] R329.25[医药卫生—中医学] R329.28R392R737.9