Protective effect of oleanolic acid on L02 hepatocyte injury induced by HgCl_2

Abstract

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.