柴胡皂苷d对H2O2诱导的肝细胞损伤的保护作用及其机制  

Protective effect of saikosaponin-d on H2O2-induced hepatocellular injury and its mechanism

作　　者：黄小凤 刘泽干 雷攀[2] 孟忠吉 王莉博[1] 杜士明 HUANG Xiao-feng;LIU Ze-gan;LEI Pan;MENG Zhong-ji;WANG Li-bo;DU Shi-ming(Department of Medicine,Hubei University of Medicine,Shiyan 442000,China;Taihe Affiliated Hospital of Hubei University of Medicine,Hubei University of Medicine,Shiyan 442000,China;Hubei Key Laboratory of Wudang Local Chinese Medicine Research,Hubei University of Medicine,Shiyan 442000,China;Institute of Biomedical Research,Hubei University of Medicine,Shiyan 442000,China)

机构地区：[1]湖北医药学院药学院,湖北十堰442000 [2]湖北医药学院附属医院十堰市太和医院,湖北十堰442000 [3]湖北医药学院武当特色中药研究湖北省重点实验室,湖北十堰442000 [4]湖北医药学院生物医学研究所,湖北十堰442000

出　　处：《中国药理学与毒理学杂志》2021年第3期169-175,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：中小企业科技创新基金(10c26214204534);武当特色中药研究湖北省重点实验室(湖北医药学院)开放课题(WDCM2019015);十堰引导性科研项目(19Y24)。

摘　　要：目的探讨柴胡皂苷d(SSd)对H_(2)O_(2)诱导L02细胞保护作用及机制。方法①L02细胞分别用SSd 0.5,1和2μmol·L^(-1)预处理6 h,加H_(2)O_(2)800μmol·L^(-1)处理24 h,同时设细胞对照和H_(2)O_(2)800μmol·L^(-1)(模型)组;MTT法检测细胞存活率,生化检测法检测细胞上清液中谷草转氨酶(GOT)和谷丙转氨酶(GPT)活性及丙二醛(MDA)和乳酸脱氢酶(LDH)含量。②L02细胞分别用SSd 2μmol·L^(-1)和特丁基对苯二酚(tBHO)50μmol·L^(-1)预处理6 h,加H_(2)O_(2)800μmol·L^(-1)处理24 h,同时设细胞对照、模型和单独SSd 2μmol·L^(-1)组;荧光探针分析法检测细胞内活性氧(ROS)积累,免疫荧光法检测核因子E2相关因子2(Nrf2)核转位水平,Western印迹法检测Nrf2和血红素氧合酶1(HO-1)蛋白表达水平。结果①MTT结果显示,与细胞对照组相比,模型组细胞存活率显著下降(P<0.05),与模型组相比,模型+SSd 1和2μmol·L^(-1)组细胞存活率显著升高(P<0.05);生化检测结果显示,与细胞对照组相比,模型组GPT和GOT活性及MDA和LDH含量均显著升高(P<0.05);与模型组相比,模型+SSd 2μmol·L^(-1)组GPT和GOT活性及MDA和LDH含量均显著降低(P<0.05)。②ROS检测结果显示,与细胞对照组相比,模型组ROS显著升高(P<0.05);与模型组相比,模型+SSd 2μmol·L^(-1)组显著降低(P<0.05);免疫荧光结果显示,模型+SSd 2μmol·L^(-1)组Nrf2主要定位于细胞核中;Western印迹结果显示,模型组细胞核及细胞质中的Nrf2蛋白及HO-1蛋白表达水平没有显著提升;与模型组相比,模型+SSd 2μmol·L^(-1)组以及模型+tBHQ 50μmol·L^(-1)组Nrf2和HO-1蛋白表达水平显著增强(均P<0.05);与细胞对照组及模型组相比,单独SSd 2μmol·L^(-1)组可以显著提高L02细胞中Nrf2和HO-1蛋白表达(P<0.05)。结论SSd能有效保护H_(2)O_(2)诱导的L02细胞损伤,其机制与降低细胞内ROS含量,上调Nrf2和HO-1蛋白表达有关。OBJECTIVE To investigate the effects of saikosaponin-d(SSd)on the proliferation of L02 cells induced by H_(2)O_(2) and the mechanism.METHODS①L02 cells were pretreated with SSd 0.5,1 and 2μmol·L^(-1) for 6 h,and administered with H_(2)O_(2)800μmol·L^(-1) for 24 h.Cell viability was analyzed by MTT assay,while the activities of glutamic-oxalacetic transaminase(GOT),glutamic-pyruvic transaminase(GPT),as well as the contents of malondialdehyde(MDA),and lactate dehydrogenase(LDH)in cell culture were detected by biochemical assay.②L02 cells were pretreated with SSd 2μmol·L^(-1) and tert-butylhydroquinone(tBHQ)50μmol·L^(-1) for 6 h,and administered with H_(2)O_(2)800μmol·L^(-1) for 24 h.The intracellular accumulation of reactive oxygen species(ROS)was analyzed by fluorescent probes,nuclear translocation of nuclear factor-erythroid 2-related factor 2(Nrf2)was determined by immunofluorescence assay,the protein expressions of Nrf2 and heme oxygenase-1(HO-1)were detected by Western blotting.RESULTS①Cell survival rate was decreased in model group significantly(vs control,P<0.05),but increased in H_(2)O_(2)+SSd 1 and 2μmol·L^(-1) group significantly(vs H_(2)O_(2),P<0.05).The activities of GPT,GOT and the contents of MDA and LDH were significantly increased in model group(P<0.05).Compared with model group,the activities of GPT,GOT and the contents of MDA and LDH were significantly decreased in H_(2)O_(2)+SSd 2μmol·L^(-1)(vs H_(2)O_(2),P<0.05).②H_(2)O_(2) significantly increased ROS production(vs cell control,P<0.05),in H_(2)O_(2)+SSd 2μmol·L^(-1) group,but the ROS levels were significantly reduced(vs H_(2)O_(2),P<0.05).Compared with the cell control group,the localization of Nrf2 in the nucleus was increased intuitively in SSd 2μmol·L^(-1) and tBHQ 50μmol·L^(-1) group.Compared with H_(2)O_(2) group,the protein expressions of Nrf2 and HO-1 in H_(2)O_(2)+SSd 2μmol·L^(-1) or H_(2)O_(2)+tBHQ 50μmol·L^(-1) group were enhanced(all P<0.05).SSd 2μmol·L^(-1) alone could significantly increase the expre

关 键 词：柴胡皂苷-D 非酒精性脂肪性肝病 核因子E2相关因子2 血红素氧合酶1 氧化应激 

分 类 号：R285[医药卫生—中药学]