Objective  To investigate the method of establishing a model of oxidative damage of L02 hepatocytes.

  Methods  L02 hepatocytes were cultured divided into 3 h damage group, 6 h group and 12 h group. Each group was divided into 6 subgroups according to different concentrations of hydrogen peroxide (H₂O₂) added: 100, 200, 300, 500, 750, 1 000 μmol/L. The control group was treated without H₂O₂. All groups were tested with cell counting kit (CCK)-8 after incubating for 3 h, 6 h or 12 h respectively. The other L02 hepatocytes were cultured and divided into 3 h damage group, 6 h group and 12 h group. The 3 h group was divided into 5 subgroups according to different concentrations of H₂O₂ added: 100, 200, 300, 500 and 750 μmol/L, the 6 h group was divided into 4 subgroups according to different concentrations of H₂O₂ added: 100, 200, 300 and 500 μmol/L, and the 12 h group was divided into 3 subgroups according to different concentrations of H₂O₂ added: 100, 200 and 300 μmol/L. The control group was treated without H₂O₂. All groups were tested by flow cytometry with Annexin V-fluorescein isothiocyanate(FITC)/propidium iodide(PI)double staining after incubating for 3 h, 6 h or 12 h respectively. The model of oxidative damage of L02 hepatocytes was established and identified. The L02 hepatocytes in damage group were treated with 200 μmol/L H₂O₂ after culture and L02 hepatocytes in control group were treated without H₂O₂ after culture. Indicators such as mitochondrial membrane potential, malondialdehyde (MDA), oxygen free radical (ROS), superoxide dismutase (SOD), alanine aminotransferase (ALT), aspartate aminotransferase (AST) were tested after L02 hepatocytes were incubated for 6 h.

  Results  Compared with control group, significant differences were observed in cell survival rates of 200, 300, 500, 750 and 1 000 μmol/L subgroup in 3 h damage group (all in P<0.01). So were in 6 h damage group and 12 h damage group (all in P<0.01). The correlation coefficient between H₂O₂ concentration and cell viability was -0.993 in 3 h group, -0.955 in 6 h group, and -0.819 in 12 h group. Flow cytometry with Annexin V-FITC/PI double staining was used to detect the apoptosis/necrosis of L02 hepatocytes when treated with different concentrations and actuation duration of H₂O₂. In 3 h, 6 h and 12 h damage group, significant differences were observed between damage subgroups and control groups(all in P<0.01). The correlation coefficients between H₂O₂ concentration and cell apoptosis/necrosis in 3 h, 6 h and 12 h group were 0.971, 0.992 and 0.986 respectively. Compared with control group, significant differences were observed in mitochondrial membrane potential, MDA, ROS, SOD, ALT, and AST in damage group after treated with 200 μmol/L H₂O₂ for 6 h(all in P<0.01).

  Conclusions  L02 hepatocytes treating with 200 μmol/L H₂O₂ for 6 h is an appropriate model simulating the ischemia-reperfusion or oxidative damage in vitro.