DNA Methylation Of The GRPR Gene In CUMS Model Of Depression

Objective:The aim of the present study was to investigate GRPR alterations in SD rat hypothalamus following stress and fluoxetine treatment,and explore DNA methylation at the GRPR gene promoter.Methods:A total of 30 male rats were randomly divided into three groups(n=10/group):the chronic unpredictable mild stress(CUMS)+ saline group(CUMS group);the CUMS+ fluoxetine group(fluoxetine group)and the control group.The rats in the CUMS group and fluoxetine group were exposed to 7 different stressors for 4 weeks:24h food deprivation;24h water deprivation;24h 45° tilted cages;damp sawdust;5min 4? ice water swimming;5min 45? hot water swimming;3min clamped tails.Then,we performed the body weight,sucrose preference test and forced swimming test(FST)to detect the effects of stress and fluoxetine on anhedonia and activity.Western blotting and real-time PCR were used to study the protein and mRNA expression levels of GRPR in the hypothalamus.In addition,Methylated DNA Immunoprecipita-tion Sequencing(MeDIP-Seq)were used to explore DNA methylation at the GRPR gene promoter.Results:(1)Sucrose preference tests,body weight test and forced swimming test:Before the CUMS procedure,no significant difference was observed among the three groups(p>0.05).At the end of CUMS procedure,the CUMS group and the fluoxetine group were significantly different from the normal control group,showing a lower index of body weight,sucrose preference and increase in the immobility time of FST,fluoxetine treatment reversed CUMS-induced depressive-like behavior(p<0.05).(2)GRPR protein expression:The GRPR protein level of CUMS group was significantly increased compared with the control group and the fluoxetine group(p<0.05),and the expression of hypothalamic GRPR protein in the control group had no significant differences with the fluoxetine group(p>0.05)(3)GRPR mRNA expression:RT-PCR analysis was performed to investigate the level of GRPR mRNA expression in the hypothalamus.The RT-PCR data revealed that mRNA expression of hypothalamic GRPR in control group had no significant differences with the fluoxetine group(p>0.05),but had significant difference with the CUMS group(p<0.05).(4)The levels of DNA methylation at the GRPR gene promoter:The levels of DNA methylation at the GRPR gene promoter in the CUMS group were significantly lower than those in the control group(p<0.05),while fluoxetine treatment reversed this changes.Conclusion:(1)The SD rat depression model established by CUMS and after treatment of fluoxetine,the rat had an improvement on depressive symptoms.(2)The results showed that the exposure of rats to CUMS can induce depressive behavior that is associated with changes of GRPR expression in the hypothalamus.And after fluoxetine treatment,the GRPR protein and mRNA expression in the hypothalamus is no significant difference with the control group.(3)Stress-induced increase of GRPR expression in the hypothalamus correlates with a specific decrease in DNA methylation of the GRPR promoter.Fluoxetine treatment reversed the CUMS-induced decrease in DNA methylation of the GRPR promoter.(4)This results may enriched the understanding of pathological process of depression and provide novel insight into therapeutic approaches to depression.