齐墩果酸对氯化汞诱导L02肝细胞损伤的保护作用  

Protective effect of oleanolic acid on L02 hepatocyte injury induced by HgCl

作　　者：李晓红[1] 刘玉梅 周健[1,2] 欧阳钏 柯鸿阳 李万伟 Li Xiaohong;Liu Yumei;Zhou Jian;Ouyang Chuan;Ke Hongyang;Li Wanwei(School of Public Health,Weifang Medical College,Weifang 261053,China;Weifang Key Laboratory of Health Inspection and Quarantine,Weifang 261053,China)

机构地区：[1]潍坊医学院公共卫生学院,维坊261053 [2]潍坊市卫生检验与检疫重点实验室,维坊261053

出　　处：《卫生研究》2021年第5期781-787,共7页Journal of Hygiene Research

基　　金：山东省自然科学基金(No.ZR2020MH336);山东省医药卫生科技发展计划项目(No.2019WS592);潍坊市科学技术发展计划(No.2019GX027)。

摘　　要：目的在明确HgCl_(2)诱导L02肝细胞损伤的基础上,探讨齐墩果酸(oleanolic acid, OA)的保护作用。方法 L02细胞按照处理方式不同分为:对照组、齐墩果酸组(OA,10μmol/L)、HgCl_(2)组(HgCl_(2),40μmol/L)及齐墩果酸+HgCl_(2)组(OA+HgCl_(2)),对照组给予无血清培养基,OA组给予OA溶液预处理8 h, HgCl_(2)组暴露于HgCl_(2)溶液6 h, OA+HgCl_(2)组先给予OA溶液处理8 h,再暴露于HgCl_(2)溶液6 h。采用MTT检测细胞活力;激光共聚焦检测JC-1探针荧光强度以确定线粒体膜电位;DCFH-DA荧光探针结合流式细胞仪检测细胞活性氧(reactive oxygen species, ROS)水平;Annexin V/PI双染法结合流式细胞仪测定细胞凋亡率;过氧化氢酶(catalase, CAT)、总超氧化物歧化酶(total superoxide dismutase, T-SOD)、谷胱甘肽(glutathione, GSH)、丙二醛(malondialdehyde, MDA)、Caspase 3和Casepase 9试剂盒结合酶标仪测定其活力及含量水平。结果与对照组相比,40μmol/L HgCl_(2)可导致细胞活力降至0.52±0.03(P<0.05),OA预处理可显著改善HgCl_(2)诱导的细胞活力降低,活力水平为0.86±0.05,较HgCl_(2)组显著升高(P<0.05);线粒体膜电位检测结果显示细胞暴露于40μmol/L HgCl_(2),红色荧光强度较对照组显著减弱,红绿荧光比值为0.23±0.02(P<0.05),OA预处理可明显增加红色荧光,红绿荧光比值为1.32±0.08,较HgCl_(2)组显著增加(P<0.05);细胞暴露于40μmo/L HgCl_(2),其ROS相对荧光强度为1.21±0.07,细胞凋亡率可达8%左右,Casepase 3及Casepase 9的活力水平分别为3.11±0.20、2.94±0.17,均较对照组显著升高(P<0.05),OA预处理可明显缓解上述指标的变化,与HgCl_(2)组相比,差异有统计学意义(P<0.05);40μmo/L HgCl_(2)处理组,T-SOD水平为(7.68±0.39)U/mL,显著低于对照组(P<0.05),MDA水平为(4.99±0.26)nmol/mg,较对照组显著升高(P<0.05);OA预处理组,T-SOD水平为(13.97±0.71)U/mL,显著高于HgCl_(2)组(P<0.05),MDA水平较HgCl_(2)组显著降低,为(3.01±0.17)nmol/mg(P<0.05)。结论OBJECTIVE To investigate the protective effect of oleanolic acid(OA) on HgCl_(2) induced liver injury. METHODS L02 cells were divided into four groups according to different treatment, control group(Con), oleanolic acid group(OA,10 μmol/L), HgCl_(2) group(HgCl_(2),40 μmol/L) and oleanolic acid + HgCl_(2) group(OA + HgCl_(2)). Cells of control group were given serum-free medium, cells of OA group were pretreated with OA solution for 8 hours, cells of HgCl_(2) group were exposed to HgCl_(2) solution for 6 hours, cells of OA + HgCl_(2) group were pretreated with OA solution for 8 hours, and then exposed to HgCl_(2) solution for 6 hours. MTT assay was used to detect cell viability. Laser confocal scanning was used to detect JC-1 probe fluorescence intensity to determine mitochondrial membrane potential. DCFH-DA fluorescence probe combined with flow cytometry was used to detect reactive oxygen species(ROS) level. Annexin V/PI double staining method combined with flow cytometry was used to determine cell apoptosis rate. Catalase(CAT), total superoxide dismutase(T-SOD), glutathione(GSH), malondialdehyde(MDA), Caspase 3 and Caspase 9 kits combined with enzyme labeled instrument were used to determine their activity or content respectively. RESULTS Compared with the control group, 40 μmol/L HgCl_(2) could significantly reduce cell viability, the level was 0.52±0.03(P<0.05), OA pretreatment could significantly inhibit the decrease of cell viability induced by HgCl_(2), the level was 0.86±0.05(P<0.05). The result of mitochondrial membrane potential detection showed that cell exposed to 40 μmol/L HgCl_(2) significantly reduced the intensity of red fluorescence, and the ratio of red to green fluorescence was 0.23±0.02(P<0.05). OA pretreatment significantly increased red fluorescence, and the ratio of red fluorescence to green fluorescence was 1.32±0.08, which was significantly higher than that of HgCl_(2)(P<0.05). After exposure to 40 μmol/L HgCl_(2), the relative fluorescence intensity of ROS was 1.21±0.07, the apo

关 键 词：齐墩果酸 氯化汞 活性氧 线粒体膜电位 细胞凋亡 

分 类 号：R286[医药卫生—中药学] R965[医药卫生—中医学]