| Objective:To investigate how the ethanol affected the lipid metabolism of liver cells and the expression of its related proteins through observing the different concentrations of ethanol on the H4-IIE cells of rats.Methods:1. The cultivation of the cell According to the cell density, take a certain amount H4-IIE cells to a clean culture bottle, adding suitable amount of DMEM broth at 37 ℃,5% CO2 incubator in training; 2. The detection of the cell viability in MTT method make a concentration gradient of ethanol in the cells, and establish the role of time for 24 h, 48 h, 72 h; then choose one activity which has a certain effect on the cell growth as experiment ethanol concentration (0.2,0.4,0.6 mol/1), the experimental time shall be set at 24 h.3. Cell morphological observation Observing the cell morphology through means as HE staining, oil red O staining, AnnexinV-FITC staining, immunohistochemical staining and inverted microscope.4. Immnunohistochemical staining:To investigate the expression of H4-IIE cells, AMPK、PPARα on the test groups, in SP kit.5. Elisa method: To detect the overflow of AMPK, ACC, CPT-I, FFA in culture supernatant of H4-IIE cells with gradient concentration ethanol in 12 h,24 h,48 h,72 h different times.6. Western blotting method:To detect the expression of P-AMPK、P-ACC、CPT-I、PPAR-a of H4-IIE cells in every group. 7. Flow cytometry To detect the apoptosis of each groups.Besults:1. MTT A certain concentration of ethanol would has a obvious inhibitory effect on the growth of H4-IIE cells which is closely related to the duration and the concentration of ethanol (P< 0.05); 2. The cell morphological observation The cells’ growth condition of control group is good, the ethanol groups showed different degree changes as the 0.6 mol/1 group apoptotic changes significantly worse than 0.2,0.4 mol/1 experimental groups; 3. Immunohistochemical staining results Compared with the control group, the expression of AMPK, PPARa of 0.2,0.4,0.6 mol/1 experimental groups were significantly decreased; the expression rate of 0.6 mol/1 ethanol group is lower than 0.4 mol/1 group, the expression rate of 0.4 mol/1 group also belower than 0.2 mol/1 group (P< 0.05); 4. Elisa results In a certain concentration range and function time, ethanol can increase the overflow of AMPK, ACC, CPT-I, FFA in culture supernatant of H4-IIE cells; when the ethanol concentration within the scope of 0.1 to 1.4 mol/1, the overflow of AMPK, CPT-1 in culture supernatant of cells increases as the concentration of the ethanol and the extension of time increasing ;and the leakage of AMPK, CPT-1 in 1.6 mol/1 group (duration of 48 h, 72 h) was less than 1.4 mol/1 group in a declining trend; 5. Western blotting results Compared with the control group, the expression of P - AMPK, P - ACC, PPARa and CPT- Ⅰ in ethanol experimental groups was significantly decreased; as the growth of the ethanol concentration, the expression of P - AMPK, P - ACC, PPARa and CPT-Ⅰ in ethanol experimental groups was gradually declined (0.6 mol/1 < 0.4 mol/1 < 0.2 mol/1) (P< 0.05). 6. Flow cytometry method (1) compared with control group, the ethanol experimental cell apoptosis rate increased significantly;And the apoptosis rate of 0.6 mol/1 group> 0.4 mol/1 > 0.2 mol/1 group (P< 0.05); (2) compared with control group, the normal living cells was significantly reduced, ethanol group and 0.6 mol/1 < 0.4 mol/1< 0.2 mol/1 group (P<0.05); (3) Compared with the control group, the ethanol group apoptotic cells and dead cells was significantly increased, and 0.6 mol/1 group> 0.4 mol/1> 0.2 mol/1 group (P< 0.05).Conclusion:1. The ethanol has inhibitory effect to the activity of liver cells, and can induce the apoptosis of liver cells.2. Alcohol can cause liver cell lipid metabolic disturbance, increase fatty acid content and inhibit the oxidation, the mechanism may be related to AMPK and its relevant signal ways. |
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