| BackgroundEthanol is a common addicted substance,chronic repeated exposure to ethanol can lead to adaptive changes in brain function.Once someone drinks less or stops drinking,he will appear withdrawal syndrome,such as irritability,aggressive,loss of appetite,and seizures,its pathogenesis is not yet clear.Studies have shown that epigenetic modification is involved in the pathological process of ethanol dependence and NMDA receptors especially NR2B are closely associated with ethanol withdrawal syndrome,but the results of its epigenetic regulation research are not unanimous.This research will discussed the relationship between NR2B expression and ethanol withdrawal syndrome from the aspects of histone acetylation.Objectives1.To establish a rat model of chronic ethanol exposure,and observe behavioral changes of chronic ethanol exposured rats at different withdrawal time.2.To detect the changes of N-methyl-D-aspartate(NMDA)receptor 2B subunit(NR2B)protein expression,NR2Bm RNA expression and the level of histone H3K9 acetylation in NR2B promoter region in the hippocampus of chronic ethanol exposured rats at different withdrawal time.3.To invesitigate the effect of epigenetic modification on ethanol withdrawal syndrome.Methods1.72 male Wistar rats were randomly divided into 6 groups: withdrawal 2h group,withdrawal 6h group,withdrawal 12 h group,withdrawal 1d group,withdrawal 3d group and control group,12 rats in each group.In the 5 withdrawal groups,ethanol was administered in drinking water at the concentration of 6%(v/v)for 16 weeks,while rats in control group were maintained with water.2.Withdrawal syndromes were evaluated at 2h,6h,12 h,1d,3d after ethanol was removed in the withdrawal groups and at 2h after water was removed in the control group.After the withdrawal symptoms were evaluated,the rats were sacrificed and the brain tissues were prepared for the following experiment.3.The expression of NR2B protein in the hippocampus were measured by immunofluorescence and western blot.4.The expression of NR2Bm RNA in the hippocampus were detected by real-time PCR.5.The levels of histone H3K9 acetylation of NR2B promoter region in the hippocampus were measured by CHIP.Results1.Ethology results : The daily average absolute ethanol consumption of ethanol-exposed rats was 7.39g/kg body weight.We did not record any obvious changes in ethanol intake during the entire experimental period.There was no significant difference in body weight between the control and ethanol fed rats either at the start of the experiment((264.00±15.20)g versus(264.40±13.15)g;P>0.05),or at the end of the experiment((454.71±14.16)g versus(447.69±56.38)g;P>0.05).During chronic ethanol exposure,rats were quiet,not easily irritated and did n ot display aggressive behavior.After ethanol delivery was discontinued,these rats e xhibited stereotypical withdrawal behaviors including agitation,hyperactivity,tail stif fness,and abnormal body posture and gait.Compared with withdrawal scores of control group(1.50±0.80),these of withdrawal 2h,6h,12 h,1d groups(10.42±2.50),(15.42±1.93),(9.25±2.01),(7.67±1.92)were obviously higher(P<0.05),the withdrawal scor es of withdrawal 6h group were the highest score,then gradually decreases,and th ere was no statistically significant difference compared with control group on withdr awal 3d(2.25±0.87)(P>0.05).2.Immunofluorescence results: Compared with the expression of NR2B fluorescence intensity in the hippocampus of control group(378.56±67.65),these of withdrawal 2h,6h,12 h,1d group(1411.36 ± 210.72),(987.38 ± 104.32),(823.62 ± 48.14),(554.71 ± 73.13)were significantly increased(P<0.05).The expression of withdrawal 3d group(437.24±123.76)were also inscreased,but there were no statistical difference compared with control group(P>0.05).3.Western blot results: Compared with the expression of NR2B protein in the hippocampus of control group(0.481 ± 0.085),these of withdrawal 2h,6h,12 h,1d group(2.201 ± 0.375),(1.535 ± 0.137),(1.117 ±0.149),(0.677 ± 0.054)were significantly increased(P<0.05).The expression of withdrawal 3d group(0.530 ± 0.113)were also inscreased,but there were no statistical difference compared with control group(P>0.05).4.Real-time PCR results: Compared with the expression of NR2Bm RNA in the hippocampus of control group(0.130 ± 0.022),these of withdrawal 2h,6h,12 h,1d group(0.330 ±0.046),(0.242 ±0.059),(0.202 ±0.044),(0.179 ±0.039)were significantly increased(P<0.05).The expression of withdrawal 3d group(0.154 ± 0.022)were also inscreased,but there were no statistical difference compared with control group(P>0.05).5.Chromatin coprecipitation results: Compared with the expression of histone H3K9 acetylation of NR2B promoter region in the hippocampus of control group(8.949±1.211),these of withdrawal 2h,6h,12 h,1d group(22.258 ± 2.951),(17.200 ± 0.890),(15.028 ±1.348),(13.572 ± 1.778)were significantly increased(P<0.05).The expression of withdrawal 3d group(9.458 ±1.402)were also inscreased,but there were no statistical difference compared with control group(P>0.05).6.The correlation analysis results: Withdrawal scores were positively correlated with the expression of NR2B protein(r=0.522,P<0.01),the expression of NR2B protein were positively correlated with the expression of NR2Bm RNA(r=0.746,P<0.01),the expression of NR2Bm RNA were positively correlated with the level of histone H3K9 acetylation in NR2B promoter region(r=0.659,P<0.01),the level of histone H3K9 acetylation in NR2B promoter region were positively correlated with withdrawal scores(r=0.479,P<0.01).Conclusions1.The expression of NR2B protein and NR2Bm RNA and the level of histone H3K9 acetylation in NR2B promoter region were up-regulation in the hippocampus after chronic alcohol exposure at different withdrawl time.2.The histone H3K9 acetylation in NR2B promoter region regulates the expression of NR2Bm RNA leading to the change of NR2B protein expression,which is likely to be one of the important mechanisms of ethanol withdrawal syndrome.BackgroundStriatum as an important part of the reward pathway,plays an important role in the change of behavioral of ethanol dependence.It has become the important brain area on the research of chronic ethanol exposure.NMDA receptor is an important target of ethanol in the central nervous system,its receptor subunit NR2B is closely associated with ethanol withdrawal syndrome.This study intends to analyze the correlation between the expression of NR2B in the striatum and the withdrawal syndrome after chronic ethanol exposure at different withdrawal time,investigating the related mechanism of ethanol withdrawal syndrome.Objectives1.To establish a rat model of chronic ethanol exposure,observe behavioral changes of chronic ethanol exposured rats at different withdrawal time.2.To detect the changes of NR2B protein expression and NR2Bm RNA expression in the striatum of chronic ethanol exposured rats at different withdrawal time.3.To investigate the mechanism of ethanol withdrawal syndrome.Methods1.72 male Wistar rats were randomly divided into 6 groups: withdrawal 2h group,withdrawal 6h group,withdrawal 12 h group,withdrawal 1d group,withdrawal 3d group and control group,12 rats in each group.In the 5 withdrawal groups,ethanol was administered in drinking water at the concentration of 6%(v/v)for 16 weeks,while rats in control group were maintained with water.2.Withdrawal syndromes were evaluated at 2h,6h,12 h,1d,3d after ethanol was removed in the withdrawal groups and at 2h after water was removed in the control group.After the withdrawal symptoms were evaluated,the rats were sacrificed and the brain tissues were prepared for the following experiment.3.The expression of NR2B protein in the striatum were detected by immunofl uorescence and western blot.4.The expression of NR2Bm RNA in the striatum were measured by real-time PCR.Results1.Ethology results:Same as the first part.2.Immunofluorescence results: Compared with the expression of NR2B fluorescence intensity in the striatum of control group(2210.00±178.20),these of withdrawal 2h,6h,12 h,1d group(2710.56 ± 194.21),(5035.16 ± 234.41),(3326.23 ± 378.16),(2570.64 ±177.88)were significantly increased(P<0.05).The expression of withdrawal 3d group(2021.10±184.43)were no statistical difference compared with control group(P>0.05).3.Western blot results: Compared with the expression of NR2B protein in the striatum of control group(0.151±0.010),these of withdrawal 2h,6h,12 h,1d group(0.186± 0.012),(1.444 ± 0.134),(0.771 ± 0.085),(0.173 ± 0.013)were significantly increased(P<0.05).The expression of withdrawal 3d group(0.148 ± 0.011)were no statistical difference compared with control group(P>0.05).4.Real-time PCR results: Compared with the expression of NR2Bm RNA in the striatum of control group(0.007±0.001),these of withdrawal 2h,6h,12 h,1d group(0.028± 0.003),(0.316 ± 0.049),(0.238 ± 0.033),(0.022 ± 0.004)were significantly increased(P<0.05).The expression of withdrawal 3 d group(0.012 ± 0.004)were no statistical difference compared with control group(P>0.05).5.The correlation analysis results: Withdrawal scores were positively correlated with the expression of NR2B protein(r=0.683,P<0.01),the expression of NR2B protein were positively correlated with the expression of NR2Bm RNA(r=0.929,P<0.01),the expression of NR2Bm RNA were positively correlated with withdrawal scores(r=0.645,P<0.01).Conclusions1.The expression of NR2B was up-regulated in the striatum of chronic ethanol exposured rats at different withdrawl time.2.The change of NR2B transcription level in the striatum of rats leading to the change of NR2B protein expression,which is likely to be one of the important mechanisms of ethanol withdrawal syndrome. |
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