三萜类化合物对化学损伤原代培养大鼠肝细胞的保护作用

Protective effects of triterpenoids on primarily cultured rat hepatocytes injured by D-galactosamine and carbon tetrachloride

目的:研究三萜类化合物对半乳糖胺(D-GalN)和四氯化碳(CCl4)损伤原代培养大鼠肝细胞的保护作用及其机制。方法:两步灌流法分离大鼠肝细胞进行原代培养,评价积雪草酸(asiaticacid,AA)和β-甘草次酸(β-glycyrrhetinicacid,GA)对D-GalN和CCl4损伤原代培养肝细胞的保护作用。光镜评价细胞生长形态,MTT法测定细胞活性,测定细胞上清天冬氨酸氨基转移酶(AST)和乳酸脱氢酶(LDH);并以荧光分光光度法测定细胞中活性氧(ROS),细胞上清活性氮终产物(NOx)和细胞内谷胱甘肽(GSH)含量;用JC-1法测定细胞线粒体膜电位(ΔΨm)。结果:AA和GA均可显著抑制D-GalN所致的AST和LDH升高(P<0.05),AA尚能提高细胞存活率(P<0.05);AA和GA也能显著抑制CCl4所致的LDH释放(P<0.05)。AA和GA均显著减少两种化学损伤细胞ROS生成和NOx释放,明显改善D-GalN所致细胞线粒体ΔΨm的降低;AA尚显著抑制两种化学损伤细胞内GSH降低。结论:三萜类化合物对D-GalN和CCl4致原代培养大鼠肝细胞损伤有保护作用,其机制与抑制细胞ROS、NOx生成和GSH降低相关,对D-GalN损伤尚有改善线粒体膜电位作用。

Objective： To investigate the protective effects and mechanism of triterpenoids on primarily cultured rat hepatocytes injured by D-galactosamine （D-GalN） or carbon tetrachloride （CCl4）. Methods ： Rat hepatocytes were isolated by two-step collagenase perfusion and cultured in RPMI 1640 medium. Protective effects of asiatic acid （AA） and β-glycyrrhetinic acid （GA） were evaluated on hepatocytes injured by D-GalN （2 mmol/L） or CCl4（10 mmol/L）. Cell morphology was observed by light microscope,cell viability was measured by MTT assay,AST and LDH were determined by an automatic analyzer. Fluorescence assay was applied to test reactive oxygen species （ROS）, nitric oxide end products （NOx） and reduced glutathione （GSH）, and JC-1 staining was used to determine rnitochondria membrane potential （△ψm）. Results. AST and LDH in medium were decreased when treated with AA and GA after D-GalN injury （P〈0. 05）, furthermore AA enhanced the hepatocyte viability （P〈0. 05）. Moreover, AA and GA significantly reduced ROS and NOx generation,and ameliorated △ψm lost induced by D-GalN. AA also inhibited GSH decrease due to D-GalN and CC14treatment. Conclusion： Both AA and GA could protect hepatocytes from D-GalN and CCl4 injuries, which is associated with reducing intracellular ROS and NOx,reversing GSH depression and ameliorating △ψm lost.