密花香薷挥发油经活性氧-线粒体途径诱导SMMC-7721细胞自噬性死亡  

Essential Oil from Elsholtizia densa Benth induces autophagic death of SMMC-7721 cells via reactive oxygen-mitochondrial pathway

作　　者：李海赢 辛文媛 任秋蓉[1] 郝俊玫 武靖松 王亚男[1] 马丹炜[1] 黄浩恒 LI Hai-ying;XIN Wen-yuan;REN Qiu-rong;HAO Jun-mei;WU Jing-song;WANG Ya-nan;MA Dan-wei;HUANG Hao-heng(College of Life Science,Sichuan Normal University,Chengdu,Sichuan 610101,China)

机构地区：[1]四川师范大学生命科学学院,四川成都610101

出　　处：《现代预防医学》2022年第19期3500-3505,共6页Modern Preventive Medicine

基　　金：四川省科技厅应用基础研究项目(2020YJ0335);2017年西南野生动植物资源保护教育部重点实验室开放基金(XNYB17-5);国家中医药管理局全国中药资源普查项目(2019PC001)。

摘　　要：目的 探讨密花香薷挥发油诱导肝癌SMMC-7721细胞死亡的作用及机理。方法 设置三个处理组,使密花香薷挥发油处理终浓度为20、40μg/ml和60μg/ml,以1%DMSO为对照。采用AO-EB染色和Annexin-V/PI双染流式细胞术分析细胞死亡率;化学荧光法检测细胞内ROS相对含量和线粒体膜电位;通过透射电子显微镜观察细胞超微结构;Western blot检测自噬相关蛋白Beclin 1和LC3的表达量;采用SPSS 17.0进行ANOVA方差分析,Duncan法进行多重比较。结果 AO/EB双染和Annexin-V/PI双染流式细胞术结果表明,随着密花香薷挥发油浓度增加,SMMC-7721细胞的死亡率增高(F=132.100,Ρ<0.001);SMMC-7721细胞内相对ROS水平升高,差异有统计学意义(F=61.580,P<0.001),而线粒体膜电位下降(F=64.300,Ρ<0.001);透射电子显微镜观察SMMC-7721细胞内出现自噬体;Western blot检测结果显示,随着密花香薷挥发油处理浓度的增加,自噬相关蛋白LC3-Ⅰ(F=504.100,Ρ<0.001)、LC3-Ⅱ(F=184.500,Ρ<0.001)和Beclin 1水平上调(F=58.690,Ρ<0.001)。结论 密花香薷挥发油可通过活性氧-线粒体途径诱导SMMC-7721细胞自噬性死亡。Objective To investigate the effect and mechanism of essential oil from Elsholtizia densa Benth on SMMC-7721 cell death. Methods The cells were divided into three treatment groups, the final concentrations of essential oil from Elsholtizia densa Benth were 20, 40 μg/ml and 60 μg/ml, and the control group was treated with 1% DMSO. The cell mortality was measured by AO-EB staining and Annexin-V/PI double staining flow cytometry. The relative ROS level and mitochondrial membrane potential in cells were detected by Chemiluminescence. Ultrastructures of cells were observed by Transmission Electron Microscope. Expression of autophagy-related proteins LC3 and Beclin 1 were assessed by western blot. SPSS17.0 was used for ANOVA analysis of variance and Duncan’s method for multiple comparisons. Result The results of AO/EB double staining and Annexin-V/PI double staining flow cytometry showed that the mortality of SMMC-7721 cells increased with the increase of the concentration of essential oil of E. densa(F=132.100, Ρ<0.001);The relative ROS level of SMMC-7721 cells increased significantly(F=61.580, P<0.001), while the mitochondrial membrane potential decreased with the increasing of essential oil(F=64.300, Ρ<0.001). Autophagosomes were observed in SMMC-7721 cells by using transmission electron microscopy. The results of Western blot showed that the levels of autophagy-related proteins LC3-Ⅰ(F=504.100, Ρ<0.001)、LC3-Ⅱ(F=184.500, Ρ<0.001)and Beclin 1(F=58.690, Ρ<0.001) were up-regulated with the increase of essential Oil. Conclusion The essential Oil from E. dense can induce autophagic death on SMMC-7721 cells through the reactive oxygen-mitochondrial pathway.

关 键 词：密花香薷 SMMC-7721细胞 自噬性死亡 活性氧 

分 类 号：R285[医药卫生—中药学]