Mechanism Of P62/Nrf2 Signaling Pathway In Alcohol-induced Liver L02 Cell Injury

Purpose: To observe the damage effect of alcohol on human liver L02 cells and the change of oxidative stress when cells were treated with different concentrations of alcohol.To explore the mechanism of p62/nrf2 signal pathway involved in this process.Methods: To detect the proliferation rate of L02 cells and the effects of various factors on the survival of L02 cells by MTT assay.Down-regulate the expression of p62 in cells by RNA interference method.The expression of p62 mRNA in L02 cells that treated by p62-siRNA was detected by RT-PCR.Western-blot was used to detect the expression of p62 protein in L02 cells.Nuclear protein and cytoplasmic protein extraction kit were used to isolate cytoplasmic and nuclear proteins,Western-blot was used to detect the expression of cytoplasmic and nuclear Nrf2 protein in L02 cells.The level of reactive oxygen species(ROS)in L02 cells that stained by DCFH-DA was observed and mensurated by fluorescence microscopy and flow cytometry respectively.The activity of glutathione peroxidase(GSH-PX)and superoxide dismutase(SOD)in L02 cells was determined by colorimetric assay and hydroxylamine method respectively.Results:1.Effects of different concentrations of Alcohol on Survival rate of liver L02 cells.Serum-free medium with alcohol concentration 50 mmol/L,100 mmol/L,200 mmol/L,400 mmol/L and 800 mmol/L,respectively,was used to treat L02 cells for 24 h,compared with 0 mmol/L alcohol group,the cell survival rates were 80.42(p<0.05)?65.09(p<0.01)?48.25(p<0.01)?41.87(p<0.01)and 37.54%(p<0.01),respectively,which showed that the cell survival rate decreased with the increase of alcohol concentration.2.Effects of p62 siRNA on the survival of L02 cells.The cell survival rate of 200 mmol/L+ p62 siRNA group was significantly lower than that of 200mmol/L alcohol treatment group(p<0.05),but the cell survival rate of 0 mmol/L+ p62 siRNA group was not significantly different from that of 0 mmol/L group.3.Effects of alcohol and p62 siRNA on reactive oxygen species in L02 cells.Observed under the fluorescent microscope of DCFH-DA staining and detected by flow cytometry,the results were as follows: The intracellular fluorescence intensity and ROS content of the 200 mmol/L alcohol group were significantly higher than that in the 0 mmol/L alcohol group(p<0.01).The intracellular ROS level in 200 mmol/L+ p62 siRNA group was significantly higher than that in 200 mmol/L alcohol group(p < 0 01).The intracellular ROS level in 0 mmol/L+ p62 siRNA group was not significantly different from that in 0 mmol/L group.4.Effects of alcohol and p62 siRNA on the expression and distribution of Nrf2 in L02 cells.The level of cytoplasmic Nrf2 in the 200 mmol/L alcohol group was significantly lower than that in the 0 mmol/L alcohol group(p<0.01),and the level of nuclear Nrf2 was increased significantly(p<0.01).The cytoplasmic Nrf2 level in 200 mmol/L + p62 siRNA group was significantly higher than that in 200mmol/L alcohol treatment group(p<0.01),the level of Nrf2 in nucleus was significantly lower than that in200 mmol/L alcohol-treated group(p<0.01).There was no significant difference in the level of Nrf2 in cytoplasm and nucleus between 0 mmol/L + p62 siRNA group and 0 mmol/L group.5.Effects of alcohol and p62 siRNA on the activities of SOD and GSH-PX in L02 cells.The activity of intracellular SOD and GSH-PX in 200 mmol/L alcohol group was significantly higher than that of the 0 mmol/L alcohol group(p<0.01).The activity of SOD and GSH-PX in 200 mmol/L+ p62 siRNA group was significantly lower than that in 200 mmol/L alcohol group(p<0.01).There was no significant difference in SOD and GSH-PX activity between 0 mmol/L+ p62 siRNA group and 0 mmol/L group.Conclusion: Alcohol can significantly reduce the survival rate of human liver L02 cells,increase the level of ROS,induce oxidative stress damage,induce the expression of antioxidant stress transcription factor Nrf2 and its nuclear translocation.This effect is regulated by p62 protein and is related to p62/Nrf2 signaling pathway.P62/Nrf2 signaling pathway has protective effect on cells.