| Objective:The objectives of this study were to clarify whether oxidative stress induced by ethanol destroys intracellular Ca2+homeostasis resulting in golgi apparatus SPCA1functional changes and whether NGF had a protective effect against ethanol.Methods1. We employed closed chamber system to mimic ethanol admiration in vitro.2. Experimental grouping was designed as follows:acute ethanol group, acute ethanol(100mM)+NAC group, acute ethanol(100mM)+NGF group.3. The neuronal viability was measured by the MTT assay, as well as the morphologic changes were studied under Light Microscopes.4. The oxidative stress was measured by flow cytometric analysis and fluorescence Microscope.5. The intracellular free Ca2+concentration was measured by multi-functional enzyme-labeling instrument6. The mRNA levels of SPCA1were tested by quantitative Real-Time polymerase chain reaction.7. The protein level of SPCA1was determined by western blotting.Result:1. After acute ethanol intake, there weren’t obvious cell morphology changes in ethanol subsets, and the optical density changes of ethanol subsets had non-statistical meaning comparing to the blank, which indicated acute ethanol intake had no effect on the viability of N2A cell.2. After acute ethanol intake, the fluorescence intensity of ethanol subsets were stronger than the blank, which were dependent of ethanol concentration with the strongest intensity at Ethanol100mM subset. In Ethanol(100mM)+NAC, the fluorescence intensity was only weaker at NAC4mM subset comparing to the ethanol intake subset.3. After acute ethanol intake, the intracellular free Ca2+concentration of ethanol subsets increased comparing to the blank, which were dependent of ethanol concentration with the highest concentration at Ethanol100mM subset. In Ethanol(100mM)+NAC, the intracellular free Ca2+concentrations decreased comparing to the ethanol intake, which were dependent of NAC concentration with the lowest concentration at NAC4mM subset. In Ethanol(100mM)+NGF, the intracellular free Ca2+concentration of NGF subsets decreased comparing to the ethanol intake subset, which were dependent of NGF concentration with the lowest concentration at NGF50ng/ml subset.4. After acute ethanol intake, the mRNA and protein levels of SPCA1of ethanol subsets increased comparing to the blank, which were dependent of ethanol concentration with the highest expression at Ethanol200mM subset. In Ethanol(100mM)+NAC, the mRNA and protein levels of SPCA1decreased comparing to the ethanol intake, which were dependent of NAC concentration with the lowest expression at NAC4mM subset. In Ethanol(100mM)+NGF, the mRNA and protein levels of SPCA1of NGF subsets increased comparing to the ethanol intake subset, which were dependent of NGF concentration with the highest expression at NGF100ng/ml subset.Conclusion:1. Acute ethanol intake induced oxidative stress, calcium overload and elevated the expression of SPCA1at mRNA and protein level in N2A cell.2. NAC pretreatment alleviated oxidative stress and calcium overload induced by acute ethanol intake and decrease the expression of SPCA1at mRNA and protein level in N2A cell.3. NGF pretreatment increased the expression of SPCA1at mRNA and protein level in N2A cell and alleviated calcium overload induced by acute ethanol intake.Figure17, table12, reference105... |
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