Li Xiaohong, Liu Yumei, Zhou Jian, Ouyang Chuan, Ke Hongyang, Li Wanwei
School of Public Health, Weifang Medical College, Weifang 261053, China.
School of Public Health, Weifang Medical College, Weifang 261053, China Weifang Key Laboratory of Health Inspection and Quarantine, Weifang 261053, China.
Wei Sheng Yan Jiu. 2021 Sep;50(5):781-787. doi: 10.19813/j.cnki.weishengyanjiu.2021.05.013.
To investigate the protective effect of oleanolic acid(OA) on HgCl_2 induced liver injury.
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.
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 apoptosis rate was about 8%, the activity levels of Casepase 3 and Casepase 9 were 3.11±0.20 and 2.94±0.17, respectively, which were all significantly higher than those in the control group(P<0.05). OA pretreatment could significantly alleviate the changes of the above indexes, and the difference was statistically significant compared with HgCl_2 group(P<0.05). The level of T-SOD in HgCl_2 group was(7.68±0.39)U/mL, which was significantly lower than that in control group(P<0.05). Compared to the control group, the level of MDA was significantly increased to(4.99±0.26)nmol/mg(P<0.05). OA pretreatment significantly increased level of T-SOD and decreased the level of MDA, the levels were(13.97±0.71)U/mL and(3.01±0.17)nmol/mg, respectively(P<0.05).
A certain concentration of HgCl_2 can induce hepatocyte damage. OA pretreatment may reduce cell damage by improving oxidative stress.
探讨齐墩果酸（OA）对氯化汞诱导的肝损伤的保护作用。
将L02细胞根据不同处理分为四组，对照组（Con）、齐墩果酸组（OA，10 μmol/L）、氯化汞组（HgCl₂，40 μmol/L）和齐墩果酸+氯化汞组（OA+HgCl₂）。对照组细胞给予无血清培养基，OA组细胞用OA溶液预处理8小时，HgCl₂组细胞暴露于HgCl₂溶液6小时，OA+HgCl₂组细胞先用OA溶液预处理8小时，然后暴露于HgCl₂溶液6小时。采用MTT法检测细胞活力。利用激光共聚焦扫描检测JC-1探针荧光强度以确定线粒体膜电位。采用DCFH-DA荧光探针结合流式细胞术检测活性氧（ROS）水平。采用Annexin V/PI双染法结合流式细胞术测定细胞凋亡率。分别使用过氧化氢酶（CAT）、总超氧化物歧化酶（T-SOD）、谷胱甘肽（GSH）、丙二醛（MDA）、半胱天冬酶3和半胱天冬酶9试剂盒结合酶标仪测定其活性或含量。
与对照组相比，40 μmol/L HgCl₂可显著降低细胞活力，水平为0.52±0.03（P<0.05），OA预处理可显著抑制HgCl₂诱导的细胞活力下降，水平为0.86±0.05（P<0.05）。线粒体膜电位检测结果显示，暴露于40 μmol/L HgCl₂的细胞显著降低了红色荧光强度，红/绿荧光比值为0.23±0.02（P<0.05）。OA预处理显著增加了红色荧光，红荧光与绿荧光比值为1.32±0.08，显著高于HgCl₂组（P<0.05）。暴露于40 μmol/L HgCl₂后，ROS的相对荧光强度为1.21±0.07，凋亡率约为8%，半胱天冬酶3和半胱天冬酶9的活性水平分别为3.11±0.20和2.94±0.17，均显著高于对照组（P<0.05）。OA预处理可显著减轻上述指标的变化，与HgCl₂组相比差异有统计学意义（P<0.05）。HgCl₂组T-SOD水平为（7.68±0.39）U/mL，显著低于对照组（P<0.05）。与对照组相比，MDA水平显著升高至（4.99±0.26）nmol/mg（P<0.05）。OA预处理显著提高了T-SOD水平并降低了MDA水平，水平分别为（13.97±0.71）U/mL和（3.01±0.17）nmol/mg（P<0.05）。
一定浓度的HgCl₂可诱导肝细胞损伤。OA预处理可能通过改善氧化应激减轻细胞损伤。
