Study Of Autophagy In Process Of Apoptosis Of SMMC-7721 Cells Induced By Ethanol

Objective: The study aims to observe the autophagy in the process of apoptosis of SMMC-7721 cells induced by ethanol by utilizing SMMC-7721 cells treated with different concentrations of ethanol.Methods: The inhibitory effect of ethanol on the proliferation of SMMC-7721 cells was analyzed with the method of MTT assay. Cell apoptosis was observed by DAPI staining. Autophagy effector protein LC3 and autophagy substrate p62 were immunostained. The ultrastructure of autophagosome was observed under the transmission electron microscope. Expressions of LC3 and p62 were assayed respectively with Western blotting.Results: Comapred with the control in serum-free medium after 24 h,ethanol at the con centration of 100 mmol/L, 200 mmol/L, 400 mmol/L and 800 mmol/L respectively reduced the proliferation of SMMC-7721 cells to 76.35%(P<0.05), 56.25%(P<0.01), 45.22%(P<0.01), 38.42%(P<0.01). That ethanol significantly inhibited the proliferation of cells in dose-dependent manner was revealed by MTT assay. Nucleus were stained by DAPI, compared with control, characteristic morphological change of apoptosis was observed with the increase of alcohol concentration. Alcohol could significantly induce the apoptosis of SMMC-7721 cells. Immunostaining has indicated that LC3 and p62 were co-localized in SMMC-7721 cellular cytosol. The expressions of LC3 and p62 were analyzed with Western blotting assay. Compared with the group in control, the expressions of LC3-II and p62 reached the peak at 2h after the SMMC-7721 cells were treated with 200mmol/L ethanol, statistically significant compared with serum free group. Though the expression of LC3-I reduced without significance. Compared with serum free group, the expression of LC3 was significantly increased and the expression of p62 was seen a significantly increase at 2h and 6h in group of 200mmol/L ethanol. When treated with serum free medium, the expression of LC3 was significantly increase, however, the trend was different from that in alcohol. The expression of p62 was seen a significantly increase at 2h and 6h while the variation was small.And the ultrastructure of autophagosome was observed under the transmission electron microscope.Conclusion: Ethanol could significantly induce the apoptosis of SMMC-7721 cells and autophagy is involved in this ethanol-inuced apoptosis of SMMC-7721 cells.