甘草抗肝窦内皮细胞缺氧损伤的活性成分筛选  

Screen active ingredient of liquorice via anti-liver sinusoidal endothelial cells hypoxia injury

作　　者：沈丽[1] 陶艳艳[1] 赵志敏[1,2] 刘洪亮[1] 刘成海[1,2,3] 

机构地区：[1]上海中医药大学附属曙光医院肝病研究所,上海201203 [2]上海市中医临床重点实验室 [3]上海高校中医内科学E-研究院

出　　处：《中西医结合肝病杂志》2015年第4期227-230,I0001,共5页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：上海市医学领军人才计划(LJ10005);上海高校中医内科学E研究院建设项目(E03008)

摘　　要：目的:采用氯化钴(Co Cl2)诱导肝窦内皮细胞SK-HEP-1缺氧模型,筛选甘草抗肝窦内皮细胞缺氧损伤的活性成分。方法:不同浓度Co Cl2(12.5-1600 M)与SK-HEP-1细胞孵育6h^72h,CCK8法与活性氧(ROS)检测评估Co Cl2诱导SK-HEP-1细胞过氧化损伤。选择800 M Co Cl2与SK-HEP-1细胞孵育24小时,同时加入不同浓度异甘草素、甘草苷、甘草素、甘草酸二铵盐、甘草酸、甘草酸单胺盐、甘草次酸(0~100 M),Ed U掺入法检测细胞增殖,细胞免疫荧光染色检测一氧化氮(NO)及一氧化氮合酶(NOS),荧光探针法检测活性氧含量。结果:与空白对照组相比,800 M Co Cl2作用24h可以显著诱导细胞缺氧并促进SK-HEP-1细胞ROS含量、抑制SK-HEP-1细胞NO含量及NOS活性,并显著抑制SK-HEP-1细胞的增殖。异甘草素、甘草酸二铵盐可显著改善Co Cl2诱导的SKHEP-1细胞缺氧及其增殖抑制作用,显著抑制NO含量及NOS,抑制ROS含量。结论:Co Cl2可诱导肝窦内皮细胞SK-HEP-1缺氧并显著抑制其增殖,甘草单体成分异甘草素、甘草酸二铵盐可以显著抑制Co Cl2诱导的肝窦内皮细胞缺氧及其增殖抑制作用。Objective：To screen the active ingredient of liquorice in hypoxia model of liver sinusoidal endothelial cells induced by cobalt chloride（Co Cl2）. Methods：SK-HEP-1 cells were incubated with different concentrations of Co Cl2（12.5-1600 M）for 6h,12 h,24h,48 h and 72 h respectively. Hypoxia injury of SK-HEP-1cells induced by 800 M Co Cl2 for 24h was evaluated by CCK8 and the content of reactive oxygen species（ROS）. Then,SK-HEP-1 cells were stimulated with 800 M Co Cl2,and treated with different concentrations of isoliquiritigenin,liquiritin,glycyrrhizin,diammonium glycyrrhizate,glycyrrhizic acid,mono-ammonium glycyrrhizinate,glycyrrhetinic acid（0-100M）for 24 h,respectively. Cell proliferation was examined with Ed U incorporation. The content of nitric oxide（NO）and ROS and activity of nitric oxide synthase（NOS）were assayed by immunofluorescent stain. Results：Hypoxia model of liver sinusoidal endothelial cells was induced by 800 M Co Cl2 for 24h accompanied with the increased expression of ROS,NO and NOS,and a decrease in cell proliferation. Hypoxia injury and inhibition of proliferation of SK-HEP-1 cells were improved by being incubated with isoliquiritigenin and diammonium glycyrrhizate. At the same time,different concentrations of isoliquiritigenin and diammonium glycyrrhizate decreased the expressions of ROS,NO and NOS in hypoxia injury of SK-HEP-1 cells. The other 5 monomer composition（liquiritin,glycyrrhizin,glycyrrhizic acid,mono ammonium isoliquiritin,glycyrrhetinic acid）did not show the similar effect. Conclusion：Co Cl2 induced the hypoxia injury of SK-HEP-1 cells and significantly inhibited proliferation of SK-HEP-1 cells. Isoliquiritigenin and diammonium glycyrrhizate significantly improved the hypoxia injury of SK-HEP-1 cells and inhibition of SK-HEP-1 cells.

关 键 词：氯化钴 肝窦内皮细胞 缺氧损伤 甘草素 甘草酸二铵盐 

分 类 号：R285[医药卫生—中药学]