湿生扁蕾乙酸乙酯提取物对乙醇诱导L02细胞氧化损伤的保护作用研究

Study on the protective effect of ethyl acetate extract of Gentianopsis paludosa on the oxidative damage of L02 cell induced by ethanol

研究湿生扁蕾乙酸乙酯提取物(GEE)对乙醇诱导人正常肝细胞(L02)氧化损伤的保护作用及其机制。用400μmol/L乙醇诱导L02细胞建立体外细胞氧化损伤模型,加入GEE 25μg/mL、50μg/mL、100μg/mL作用24 h,采用MTT法检测细胞存活率;检测细胞培养上清液中谷丙转氨酶(ALT)、谷草转氨酶(AST)、细胞内超氧化物歧化酶(SOD)和丙二醛(MDA)含量的变化;DCFH-DA探针染色流式细胞仪检测细胞内ROS水平;DTNB-GR动力学循环法检测胞内还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)的含量变化。结果表明,400μg/mL乙醇诱导L02细胞24 h可建立肝细胞氧化损伤模型。GEE在25~100μg/mL对L02细胞无损伤。与模型组比较,GEE中剂量、高剂量组能显著提高L02细胞存活率(P<0.001);降低细胞上清液中ALT、AST含量(P<0.001);使细胞内SOD活力显著提高(P<0.001);MDA含量明显降低(P<0.001);同时使细胞内ROS水平降低(P<0.01)、GSH/GSSG比值提高(P<0.001)。提示GEE对乙醇诱导的L02细胞损伤具有保护作用,其机制可能与其减轻氧化应激反应有关。

To investigate the protective effect and its mechanism of ethyl acetate extract(GEE)of Gentianopsis paludosa on the oxidative damage of human normal hepatocyte(L02)cells induced by ethanol.L02 cells induced by 400μmol·L^-1 ethanol to establish oxidative damage model in vitro.After adding 25μg·mL^-1,50μg·mL^-1,100μg·mL^-1 GEE for 24 h,the cell survival rate was measured by MTT method;the kit was used to detect changes in ALT,AST in the cell culture supernatant and SOD and MDA contents in cells;DCFH-DA probe staining flow cytometry was used to detect intracellular ROS level;DTNB-GR kinetic cycling method was used to detect intracellular GSH and GSSG content changes.L02 cells induced by400μmol·L^-1 ethanol for 24 h can be used to establish a model of oxidative damage to liver cells.Between 25~100μg·mL^-1 GEE had no damage to L02 cells.Compared with the model group,the GEE medium and high dose groups can significantly improve the survival rate of L02 cells(P<0.001);reduce the ALT and AST content in the cell supernatant(P<0.001);then significantly increase the SOD activity in the cells(P<0.001);MDA content was significantly reduced(P<0.001);at the same time,intracellular ROS levels were reduced(P<0.01),and GSH/GSSG ratio was increased(P<0.001).GEE has protective effect on ethanol-induced L02 cell damage,and its mechanism may be related to its reduction of oxidative stress response.