| BackgroundChronic ethanol exposure can lead to NMDA receptors especially NR2 B subunits up-regulate express.Some studies suggest NMDA receptors especially NR2 B subunits are associated with a variety of ethanol-associated phenotypes,NMDA receptor antagonist AP-5 inhibiting the formation of ethanol conditioned place preference(CPP),but the underlying mechanisms have not been fully elucidated.Epigenetic modifications mediating environmental factors and genome lead to gene expression change and are involved in the pathological process of complex diseases including ethanol dependence.Epigenetic mechanisms studies of NR2 B gene expression are discrepancy.To investigate the effects of sodium butyrate on ethanol-seeking behavior and H3K9 acetylation of the NR2 B gene in hippocampus of rats by ethanol,and to explore the possible mechanism of histone modification underlying the ethanol-seeking behavior or ethanol dependence.Objectives1.To establish ethanol-induced CPP and sodium butyrate intervention model in rats,and to explore the effect of sodium butyrate on ethanol dependence.2.To explore the possible mechanism of histone modification due to alcohol affecting the expression of NR2 B protein,NR2 BmRNA and H3K9 acetylation in NR2 B promoter region in the hippocampus of Wistar rats in each group.3.To explore the possible mechanism of histone modification underlying the ethanol dependence.Methods1.80 male Wistar rats were through by CPP experiment no significant 48 rats,which were randomly divided into four groups according to random number table: saline group,sodium butyrate group,ethanol group,sodium butyrate + ethanol group,with 12 rats in each group.These groups were treated with saline,ethanol(0.5 g/kg per day,10%v /v),sodium butyrate(200 mg/kg per day)and sodium butyrate(200 mg/kg per day)+ ethanol(0.5 g/kg per day,10%v /v)via intraperitoneal injections respectively.CPP was used to evaluate the ethanol-seeking behavior.2.After CPP,the rats were sacrificed and the hippocampus tissues were prepared for the following experiments.Using Western-blot,real-time PCR(RT-PCR)and chromatin immunoprecipitation(ChIP)assays,the expression of NR2 B protein,NR2 BmRNA and H3K9 acetylation in NR2 B promoter region in hippocampus were determined respectively.Results1.CPP test results:The CPP test values and CPP scores in each group were different(F=25.731,20.065,both P<0.05).CPP test values and CPP base values were analysed by matching t test in each group:CPP test values of ethanol group and ethanol+sodium butyrate group were significantly increased compared to CPP base values(t=5.749,12.621,both p<0.05).CPP test values of saline group and sodium butyrate group were not significant compared to CPP base values(t=1.776,0.346,both P>0.05).The CPP test values and CPP scores of ethanol group and sodium butyrate + ethanol group were significantly increased compared with that of the saline group(both P<0.05).The CPP test values and CPP scores of sodium butyrate group were not significant compared with that of the saline group(both P>0.05).The CPP test values of sodium butyrate + ethanol group was significantly increased compared with that of the ethanol group(P<0.05).The CPP scores of ethanol +sodium butyrate group was not significantly different compared with that of the ethanol group(P>0.05).2.Western-blot results: The expression of NR2 B protein in hippocampus in each group were different(F=12.384,P < 0.05).The expression of NR2 B protein in hippocampus of ethanol group and sodium butyrate + ethanol group were significanty increased compared with that of the saline group(both P<0.05).The expression of NR2 B protein in hippocampus of sodium butyrate group was not significant compared with that of the saline group(P>0.05).The expression of NR2 B protein in hippocampus of sodium butyrate + ethanol group was significantly increased compared with that of the ethanol group(P<0.05).4.RT-PCR results: The expression of NR2 BmRNA in hippocampus in each group were different(F=18.524,P<0.05).The expression of NR2 BmRNA in hippocampus of ethanol group and sodium butyrate + ethanol group were significantly increased compared with that of the saline group(both P < 0.05).The expression of NR2 BmRNA in hippocampus of sodium butyrate group was not significant compared with that of the saline group(P>0.05).The expression of NR2 BmRNA in hippocampus of sodium butyrate + ethanol group was significantly increased compared with that of the ethanol group(P<0.05).5.ChIP results: The expression of H3K9 acetylation in NR2 B promoter region in hippocampus in each group were different(F= 22.754,P<0.05).The expression of H3K9 acetylation in NR2 B promoter region in hippocampus of ethanol group and sodium butyrate + ethanol group were significantly increased compared with that of the saline group(both P<0.05).The expression of H3K9 acetylation in NR2 B promoter region in hippocampus of sodium butyrate group was not significant compared with that of the saline group(P>0.05).The expression of H3K9 acetylation in NR2 B promoter region in hippocampus of sodium butyrate + ethanol group was significantly increased compared with that of the ethanol group(P<0.05).6.The correlation analysis results: The CPP scores were positively correlated with the expression of NR2 B protein(r=0.474,P<0.05),with a positive correlation between the NR2 B protein expression and the NR2 BmRNA level(r=0.468,P<0.05),Meanwhile,the expression of NR2 BmRNA was positively correlated with the expression of H3K9 acetylation in NR2 B promoter region(r=0.596,P<0.05),and The CPP scores were positively correlated with the expression of H3K9 acetylation in NR2 B promoter region(r=0.542,P<0.05).Conclusions1.The ethalol-induced CPP and the expression of NR2 B gene was enhanced by sodium butyrate,the HDAC inhibitor.2.Chronic ethanol exposure upregulated the expression of NR2 B gene in hippocampus of Wistar rats,and the expression of CPP scores were positively correlated with the expression of NR2 B protein,the expression of NR2 B protein were positively correlated with the expression of NR2 BmRNA,which suggests the expression of NR2 B gene in hippocampus is likely to be one of the mechanisms underlying ethanol-seeking behavior.3.Chronic ethanol exposure upregulated the expression of H3K9 acetylation in NR2 B promoter region in hippocampus of Wistar rats,and the expression of NR2 BmRNA were positively correlated with the expression of H3K9 acetylation in NR2 B promoter region,while the expression of CPP scores were positively correlated with the expression of H3K9 acetylation in NR2 B promoter region,which suggests that the expression of H3K9 acetylation in NR2 B promoter region in hippocampus is likely to be one of the mechanisms of the expression of NR2 B gene,and may participate in the mechanism of histone modification of ethanol-seeking behavior. |
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