Study On The Changes Of Calcium Ion Concentration During Endoplasmic Reticulum Stress And Apoptosis Of Liver H4-ⅡE Tumor Cells Induced By Ethanol

Objective:To explore the mechanisms of liver injury caused by ethanol,observe the effects of different concentrations of ethanol on the inhibition of H4-ⅡE cell proliferation,changes in endoplasmic reticulum stress-related protein content as the ethanol concentration changes,changes in calcium ion concentration and cell apoptosis.Methods:1.Treat the rat H4-ⅡE cells with different concentrations of ethanol and different time of stimulation,12h,24h and 48h.The inhibitory effects on the proliferation of H4-ⅡE cells,stimulated with different concentrations of ethanol and different time,were measured by MTT assay.The rat H4-ⅡE cells were divided into The control group and the experimental group（0.05mol/L,0.1mol/L,0.2mol/L,0.4mol/L,0.8mol/L）;2.Using Fluo-3AM determined the change of intracellular calcium ion concentration after stimulating rat H4-ⅡE cells for 12h with different ethanol concentrations（0.05mol/L,0.1mol/L,0.2mol/L）;3.Using Western Blotting detected Caspase-3,Caspase-7,and Caspase-12 expressed by H4-ⅡE cells,which were treated with different concentrations of ethanol for 12 hours;4.Apoptotic cell rate were conducted by flow cytometry assay.The experimental values were analyzed by SPSS and expressed in meanstandard deviation（SD）.A single-factor multi-level variance analysis was used between each group,P values of less than 0.05 are considered statistically significant.Results:1.MTT method to detect the inhibitory effect of ethanol on H4-ⅡE cellsCompared with the control group,the cell growth was significantly inhibited treated in different ethanol concentrations（0.5mol/l、0.1mol/L、0.2mol/L）for 12h、24、48h,respectively,indicating that ethanol can inhibit the growth of H4-ⅡE cells and was positively correlated with ethanol concentration and the time of stimulation（P<0.05）.2.Fluo-3AM method for detecting intracellular calcium ion changes in H4-ⅡE cells stimulated by different concentrations of ethanolThe intracellular Ca²⁺concentration was detected in H4-ⅡE cells treated with different ethanol concentrations（0.05mol/L,0.1mol/L,0.2mol/L）for 12hours.The results showed that:as the ethanol concentration increased,the intracellular calcium ion concentration With the increase,the analysis of the difference between each group and the control group shows that the 0.2mol/L group and the control group are statistically significant（P<0.05）.3.Observe the protein expression of Casapase-3,Caspase-7 and Caspase-12in cells after 12 hours of different ethanol concentrationsExtract the H4-ⅡE intracellular protein after 12 hours of pretreatment with different doses of ethanol.After quantitative analysis of the protein,detect Caspase-3,Caspase-7,and Caspase-12 in each group of samples by Western Blotting.The results show:The expression of Caspase-3 protein treated with different concentrations of ethanol increased with the increase of ethanol concentration,and the difference between the control group and other groups was statistically significant（P<0.05）;the expression of Caspase-7 treated with different concentrations of ethanol The protein expression increased with the increase of ethanol concentration,and the differences between the groups were statistically significant（P<0.05）;the protein expression of Caspase-12 treated with different concentrations of ethanol increased with the increase of ethanol concentration,which was comparable to the control group.Compared with,0.1mol/L and 0.2mol/L histone expression increased,and the results were statistically significant（P<0.05）.4.Observation of apoptosis rate by flow cytometryThe proportion of normal viable cells in the test group（0.05mol/L、0.1mol/L、0.2mol/L）was lower than that in the control group（P<0.05）,and the proportion of early apoptosis,late apoptosis and dead cells increased（P<0.05）.The apoptotic rate showed an increasing trend with the increase of ethanol concentration.Conclusion:1.Ethanol has an inhibitory effect on the growth of rat hepatocytes,resulting in an imbalance of calcium ion homeostasis in hepatocytes and endoplasmic reticulum stress,which is related to the concentration of ethanol and the duration of action of ethanol;2.Ethanol induces the abnormal expression of endoplasmic reticulum stress and apoptosis-related proteins Caspase-3,Caspase-7 and Caspase-12,leading to cell apoptosis and causing liver cell damage;3.Alcohol-induced liver cell damage may be related to the endoplasmic reticulum stress caused by the imbalance of calcium ion homeostasis.By regulating the intracellular calcium ion concentration,it may have a preventive and therapeutic effect on alcoholic liver injury.