Study Of Nrf2 Pathway And Injury Of L02 Cells Induced By Alcohol

Objective: To investigate the effect of alcohol on human liver L02 cells and the change of Nrf2 pathway and p62 in the process,and to analyze the role of Nrf2 pathway and the relation between Nrf2 and p62 induced by alcohol.Methods: The rate of L02 cells proliferation induced by different concentrations of alcohol was analyzed by MTT assay,DAPI staining was used to test the cell apoptosis by alcohol.The intracellular level of ROS was observed by DCFH-DA staining,and FCM(flow cytometry)was used to detect the percentage of ROS in L02 cells after alcohol treatment.The changes of superoxide dismutase and glutathione peroxidase in L02 cells induced by alcohol were determined by WST-1 and colorimetry respectively.The effect of alcohol on p62 protein and intracellular cytoplasm and nucleus of Nrf2 was detected by IIF(indirect immunofluorescence)assay and Western blotting.Results: When the concentrations of alcohol were 100 mmol/L,200 mmol/L,400 mmol/L and 800 mmol/L respectively in Serum-free medium,with the control group of no alcohol,the rates of different groups liver L02 cells proliferation at 24 h were 63.04 %(P<0.01),54.26 %(P<0.01),28.55 %(P<0.01)and 19.36 %(P<0.01)by comparison with 0 mmol/L group,which showed that alcohol could significantly inhibit the proliferation of L02 cells.The influence of alcohol on the nucleus of L02 cells were observed by DAPI staining,characteristic morphological changes of apoptosis were emerging with the increaseing concentration of alcohol.Intracellular reactive oxygen species were showed by stained with DCFH-DA under optical microscope and by Flow cytometry.When the alcohol concentrations were 100 mmol /L,200 mmol/L and 400 mmol/L,the color ratioes of ROS were increased with comparison with 0 mmol/L group.At the same time,flow cytometry was used to detect the changes of ROS in L02 cells treated with 200 mmol/l alcohol for different time.The results showed that the content of ROS decreased significantly with increasing treatment time of alcohol.The changes of p62 protein and Nrf2 in L02 cells induced by alcohol were observed by indirect immunofluorescence,which showed that the level of p62 protein and Nrf2 in nucleus rised significantly with the increased concentration of alcohol.The expression of p62 and Nrf2 were also analyzed by Western blotting,the expression of p62 and Nrf2 in nucleus were rised with increasing does and time of alcohol.However,the expression of Nrf2 in cytoplasm was decreased.The activity of glutathione peroxidase was measured by colorimetry,when ethanol were at the concerntration of 100 mmol/L,200 mmol/L and 400 mmol/L,the activities of glutathione peroxidase were 2411.18 U(P<0.05),3361.57 U(P<0.01),and 3636.08 U(P<0.01).When treatment time were 3 h,6 h,12 h,and 24 h,the activities of glutathione peroxidase were 2453.19 U(P<0.01),2917.22 U(P<0.01),3195.81 U(P<0.01),and 3360.42 U(P<0.01).The activity of superoxide dismutase was measured by WST-1,when ethanol were at the concerntration of 100 mmol/L,200 mmol/Land 400 mmol/L,the activities of superoxide dismutase were 129.02 u/mgprot(P<0.05),141.32 u/mgprot(P<0.01),and 160.51 u/mgprot(P<0.01).When treatment time were 3 h,6 h,12 h and 24 h,the activities of superoxide dismutase were 128.37 u/mgprot(P<0.01),141.43 u/mgprot(P<0.01),159.90 u/mgprot(P<0.01),and 192.24 u/mgprot(P<0.01).Conclusion: Alcohol could significantly inhibit the proliferation and induce injury of L02 cells,due to raised ROS.The expression of Nrf2 in nucleus and p62 protein increased in the process of cell injury,causing an increase in the activities of superoxide dismutase and glutathione peroxidase,which had protective effect against alcohol in human liver L02 cells.