去氢木香内酯激活凋亡与自噬抑制人肺癌A549细胞生长  

Dehydrocostus Lactone Inhibits Growth of Human Lung Cancer A549 Cells Through Activation of Apoptosis and Autophagy

作　　者：田颖颖 李依林 田时秋 裴海鸾 于尚玥 王子健 左泽平 王志斌[1,2] TIAN Yingying;LI Yilin;TIAN Shiqiu;PEI Hailuan;YU Shangyue;WANG Zijian;ZUO Zeping;WANG Zhibin(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China;Research Institute of Beijing Tongrentang Co.Ltd.,Beijing 100079,China)

机构地区：[1]北京中医药大学中药学院,北京100029 [2]北京同仁堂股份有限公司科学研究所,北京100079

出　　处：《中国实验方剂学杂志》2023年第2期73-80,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家科技重大专项(2018ZX09201-011,2018ZX09301-011-003)。

摘　　要：目的:研究去氢木香内酯(DL)对人肺癌细胞A549增殖、凋亡、自噬的影响,以期阐明其作用机制。方法:采用细胞增殖与活性检测法(CCK-8)研究0、5、10、15、20、25μmol·L^(-1)DL对人肺癌A549细胞增殖能力的影响。通过细胞克隆形成实验研究DL对A549细胞克隆形成能力的影响。选择10、20μmol·L^(-1)作为DL低、高浓度组,采用荧光染料Hoechst 33258染色及蛋白免疫印迹法(Western blot)观察DL对A549细胞凋亡的影响。通过吖啶橙染色检测DL给药后自噬溶酶体的变化,利用免疫荧光实验和Western blot检测DL对微管相关蛋白1轻链3(LC3)表达水平的影响,通过对比DL单独给药及其分别与自噬抑制剂巴佛洛霉素-A1(BAF-A1)、3-甲基腺嘌呤(3-MA)联合用药对A549细胞自噬的影响。通过Western blot观察DL对A549细胞信号通路调控的作用。结果:与空白组比较,10、15、20、25μmol·L^(-1)DL组A549细胞存活率均显著降低(P<0.01);5μmol·L^(-1)DL可显著抑制A549克隆细胞形成(P<0.01),说明DL能够抑制人肺癌A549细胞的增殖能力。与空白组比较,DL组(10、20μmol·L^(-1))凋亡细胞数目增多,DL组(20μmol·L^(-1))中凋亡相关蛋白聚腺苷酸二磷酸核糖基聚合酶(PARP)和B细胞淋巴瘤-2(Bcl-2)相关X蛋白(Bax)表达明显上升(P<0.05,P<0.01),Bcl-2表达显著下降(P<0.01)。吖啶橙染色结果显示,与空白组比较,DL组(20μmol·L^(-1))橙色荧光增强,说明自噬溶酶体的生成数量增多。DL组(20μmol·L^(-1))还能使细胞内LC3橙色荧光颗粒明显增高,LC3Ⅱ的表达水平显著上升(P<0.01),且加入自噬抑制剂后,A549细胞对DL作用的敏感性显著下降(P<0.01),表明自噬参与了DL诱导的A549细胞死亡。与空白组比较,DL组(20μmol·L^(-1))能够使自噬相关蛋白3(Atg3)、自噬相关蛋白5(Atg5)的表达增加并使蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)、信号传导与转录活化因子3(STAT3)的磷酸化水平下降(P<0.05,P<0.01)。结Objective:To evaluate the effects of dehydrocostus lactone(DL)on the proliferation,apoptosis,and autophagy of human lung cancer cell A549 and to elucidate its related mechanism.Method:The effect of DL with different concentrations(0,5,10,15,20,25μmol·L^(-1))on the proliferation of human lung cancer A549 cells was investigated by cell counting kit-8(CCK-8),and its impact on the clonogenic ability of A549 cells was studied by cell clonogenic assay.The concentrations 10,20μmol·L^(-1)were selected as DL lowdose group and high-dose group.Hoechst 33258 staining and western blot were used to observe the effect of DL on apoptosis of A549 cells.Autolysosomes were detected by acridine orange staining,and the expression level of microtubule-associated protein 1 light chain 3(LC3)was determined by immunofluorescence and western blot.In addition,the effects of DL in combination with autophagy inhibitors bafilomycin A1(BAF-A1)or 3-methyladenine(3-MA)on the autophagy of A549 cells was checked by CCK-8 assay.Finally,the role of DL in the regulation of A549 cell signaling pathway was explored by Western blot.Result:Compared with the conditions in the control group,the survival rate of A549 cells in the DL groups(10,15,20,25μmol·L^(-1))was decreased(P<0.01),and 5μmol·L^(-1)DL could inhibited the formation of A549 clone cells(P<0.01),indicating that DL could inhibit the proliferation of human lung cancer A549 cells.The number of apoptotic cells was higher in both DL low-dose and high-dose groups than that in the control group,and the expression of apoptosis-related proteins poly(ADP ribose)polymerase(PARP)and B lymphocytoma-2(Bcl-2)-associated X protein(Bax)were up-regulated(P<0.05,P<0.01),while the expression of Bcl-2 was down-regulated(P<0.01)in DL highdose group.The acridine orange staining showed that the orange fluorescence in the DL high-dose group was enhanced compared with that in the control group,indicating that DL could dramatically promote the formation of autolysosomes.Moreover,20μmol·L^(-1)DL could increase

关 键 词：去氢木香内酯 肺癌 凋亡 自噬 蛋白激酶B/哺乳动物雷帕霉素靶蛋白/信号传导与转录活化因子3(Akt/mTOR/STAT3)信号通路 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学] R2-031R285.5