| Objective:To observe proliferation inhibition, the change of enzyme, lipid metabolism and apoptosis on rat hepatoma cell lines H4-â…¡E induced by ethanol with different concentration, explore the mechanism in alcoholic liver injury.Methods:The rat hepatoma cell lines H4-â…¡E was cultured in vitro. The CCK8method was adopted in measuring the proliferation inhibition of different concentration ethanol for12h,24h,48h, setting up the control group and experimental group. Cell morphology of H4-â…¡E was observed by inverted microscope, optical microscope and fluorescent microscope. The expression of ADH, ALDH, AMPK, PPARa, caspase3, caspase8, caspase9, TNF-a were detected by western blot method. Using ELISA to test the spillage of ADH, ALDH, and the apoptosis was analysised by flow cytometry.Results:1. CCK8method revealed that ethanol could inhibit H4-â…¡E proliferation in a concentration dependency.2. The typical morphological changes of the apoptosis H4-â…¡E was appeared after using ethanol.3. Western blot:(1) compared with the control group, the expression of ADH, ALDH, AMPK, PPARa had no significant change in0.05mol/L ethanol group (P>0.05), and the others were obviously reduced (P<0.05).(2) compared with the control group, the expression of caspase3, caspase8, caspase9, TNF-a in every ethanol groups were increased (P<0.05).4. ELISA:(1)12h,24h:compared with the control group, the spillage of ADH and ALDH on every concentration of ethanol groups were increased, which had statistically significant difference (P<0.05). With the increasing concentration of ethanol, the spillage was increased.(2)48h:compared with the control group, the spillage of ADH and ALDH on every concentration of ethanol groups were increased, which had statistically significant difference (P<0.05). Compared with the1.2mol/L ethanol group, the spillage of ADH and ALDH in1.6mol/L ethanol group was reduced (P<0.05).(3)72h:compared with the control group, the spillage of ADH and ALDH on every concentration of ethanol groups were increased, which had statistically significant difference (P<0.05). Compared with the1.0mol/L ethanol group, the spillage of ADH, ALDH in1.2mol/L and1.6mol/L ethanol group were reduced (P<0.05). The spillage of ADH had no significant difference between1.2mol/L and1.6mol/L ethanol group (P>0.05). The spillage of ALDH had no significant difference between1.Omol/L and1.2mol/L ethanol group (P>0.05). Compared with the1.2mol/L ethanol group, the spillage of ALDH in1.6mol/L ethanol group was reduced (P<0.05). The comparison of other groups had difference (P<0.05), with the increasing concentration of ethanol, the spillage of ADH,ALDH was increased(P<0.05).5. Flow cytometry:compared with the control group, the counts of living cells were reduced in the ethanol groups (P<0.05), the counts of early apoptotic, late apoptotic and dead cells in ethanol group were increased (P<0.05). With the increasing concentration of ethanol, the apoptosis rate had an increasing trend.Conclusion:1.ethanol inducing apoptosis on hepatocytes may be related to the abnormal metabolic pathways of ADH and ALDH.2. the reducing expression of ADH and ALDH may down-regulate the expression of AMPK and PPARa on H4-IIE cells leading injury in liver cells. |
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