Study Of Apoptosis And Autophagy Of L02 Cells Induced By Ethanol

Purpose: This study tested the process of cell apoptosis and autophagy with hepatic L02 cells.To investigate the correlation between autophagy and apoptosis,the study treated L02 cells with different concerntration of ethanol.To research the act of autophagy during the process of cell apoptosis,Beclin1-siRNA designed and tested for RNAi cells autophagy.Methods: The rate of cells proliferation was analyzed by MTT assay.The rate of cell apoptosis was tested with PI staining and Annexin V-FITC/PI dual staining.Cell apoptosis was observed by DAPI staining.Autophagolysosome was stained by acid orange(AO).Autophagy related protein LC3 were immunostained.The expression of Beclin1,LC3 and P62 was tested by Western blotting.The expression of Beclin1 mRNA was assayed by RT-PCR and agarose gelelectrophoresis.Then the change of rate was tested in cells proliferation and cell apoptosis when the cell's autophagy was depressed.Results: Ethanol at the concerntration of 50 mmol/L,100 mmol/L,200 mmol/L,400 mmol/L and800 mmol/L,the rates of different groups cells proliferation at 12 h were 92.36%,87.91%,73.27%(P<0.05),67.72%(P<0.05),51.33%(P<0.01)and at 24 h were 74.85%(P<0.05),67.40%(P<0.01),47.42%(P<0.01),42.35%(P<0.01),38.59%(P<0.01).The cell nucleus was stained by DAPI,characteristic morphological change of apoptosis was observed with the increaseing of ethanol concentration and time.The rates of different groups cells apoptosis was tested with Annexin V-FITC/PI dual staining,ethanol at the concerntration of 100 mmol/L,200 mmol/L,400 mmol/L and 800 mmol/L,the rates of different groups cells apoptosis were 13.46%(P<0.05),23.28%(P<0.01),24.95%(P<0.01)and17.31%(P<0.05).The rates of different groups cells apoptosis were 11.19%(P<0.05),16.32%(P<0.05),37.95%(P<0.01),42.37%(P<0.01)and 22.59%(P<0.01),which ethanol at the concerntration of 50mmol/L,100 mmol/L,200 mmol/L,400 mmol/L and 800 mmol/L was tested by PI staining.It was obvious that the rates of different groups cells apoptosis grown with the gradually increased concerntration of alcohol.It illustrated that alcohol could significantly induce the apoptosis of L02 cells.Ethanol at the does of 100 mmol/L,200 mmol/L,400 mmol/L and 800 mmol/L,the rates of different groups coloration with acid orange was 13.86%(P<0.01),16.70%(P<0.01),17.63%(P<0.01)and 23.11%(P<0.01).The expression of Beclin1,LC3 and p62 was analyzed by Western blotting.The expression of Beclin1,LC3 and p62 was rised with the does of alcohol which treated with cells for two hours.After treated with Beclin1-siRNA,the proteins of Beclin1,LC3 and P62 were decreased significantly.The rate of cell apoptosis then induced with alcohol increased significantly after treated cells with siRNA.Conclusion: Ethanol could significantly induce the apoptosis of L02 cells,and autophagy is related to ethanol-induced injury on L02 cells.Cells autophagy could prevent L02 cells from ethanol-inuced injury.