| Objective: Study the effects and mechanisms of an enriched environment and folic acid on the behaviors on maternal separation mice.Methods: Newborn mice were divided into 4 groups,which were control group,model group,enriched environment(EE)group and folic acid(FA)group.The model group,the EE group and the FA group were given maternal separation for 4hours every day for 28 days.At 14 days of separation,mice in each group except the control group were given distinct treatment for 28 days.The treatment was as follows: the model group mice were treated with injected saline via ip.(equal volume of folic acid),the EE group mice were treated with an enriched environment for 4hours per day,the FA group mice were injected folic acid via ip.(5 mg/kg)every day.At 10 weeks,behaviors were performed by the elevated plus maze test,open field test,novel object recognition test,forced swim test and the body weight changes were observed.After behavioral tests,hippocampus,prefrontal cortex and serum was taken.The qPCR was used to detect the mRNA levels of glucocorticoid receptor(GR),mineralocorticoid receptor(MR),11β-hydroxysteroid dehydrogenase type 1(11β-HSD1),brain derived neurotrophic factor(BDNF),corticosterone(CORT)in hippocampus and prefrontal cortex.ELISA was used to detect content of corticotropin releasing hormone(CRH),adrenocorticotropic hormone(ACTH),corticosterone(CORT)in hippocampus and serum and detect content of 5-methylcytosine(5-mC)and 5-hydroxymethylcytosine(5-hmC)in the hippocampus and prefrontal cortex.Western-Blot was used to detect protein expression levels of GR,MR,11β-HSD1 and BDNF in the hippocampus and prefrontal cortex.Results: Compared with the control group,the body weight of the mice in the model group increased initially and decreased afterwards(p<0.05),and the body weight was significantly decreased after mice treated with EE or FA(p<0.05).Activity in the elevated plus maze test was significantly improved after model mice treated with EE(p<0.05 or p<0.001).The percentage of residence time in the open arm was increased(p<0.001)but not increased in other activities after mice treated with FA(p<0.05).The activity in the open field was significantly improved after model mice treated with EE(p<0.05 or p<0.001),the percentage of residence time in field center was increased(p<0.001)but not increased in other activities after mice treated with FA(p>0.05).In the forced swim test,the immobility time of the model group mice was significantly prolonged(p<0.01),and mice treated with EE or FA could reduce the immobility time dramatically(p<0.01 or p<0.001).In the novel object recognition experiment,compared with the control group,mice treated with EE or FA could improve the rate of novel object recognition drastically(p<0.01 or p<0.001).The data of qPCR shown the mRNA levels of GR,MR and BDNF in the hippocampus and prefrontal cortex were markedly improved after model mice treated with EE or FA(p<0.05 or p<0.01).The mRNA levels of CORT in the hippocampus and prefrontal cortex were obviously reduced after model mice treated with EE or FA(p<0.05 or p<0.01 or p<0.001).The mRNA levels of 11β-HSD1 in the hippocampus were clearly upgraded after model mice treated with EE or FA(p<0.05 or p<0.001),and the mRNA levels of 11β-HSD1 in the prefrontal cortex were significantly decreased after model mice treated with EE or FA(p<0.01 or p<0.001).ELISA results were shown that the content of CRH,ACTH and CORT in the hippocampus was markedly decreased after model mice treated with EE or FA(p<0.05 or p<0.01 or p<0.001).The content of ACTH in the serum in mode,EE and FA group was decreased significantly(p<0.001 or p<0.05 or p<0.01).Content of CRH and CORT in the serum addition was significantly decreased after model mice treated with EE(p<0.05 or p<0.001),mice treated with FA could only reduce the content of CORT in serum(p<0.01).The content of 5-mC in the hippocampus was considerably ameliorated after model mice treated with EE or FA(p<0.05 or p<0.01),and treated model group mice with EE or FA also could drop the content of 5-hmC in hippocampus.However,in the prefrontal cortex,only the 5-mC content in the prefrontal cortex of the EE group was increased(p<0.01)and the 5-hmC content in the FA group was decreased(p<0.001)while there was no statistical significance inother groups(p>0.05).Meanwhile,Western-Blot results showed that the protein expression levels of GR,11β-HSD1 and BDNF in the hippocampus were significantly improved after model mice treated with EE or FA(p<0.05 or p<0.01).There was no statistical significance in MR protein in hippocampus of each group(p>0.05).Compared with the control group,the ratio of GR/MR in the hippocampus and prefrontal cortex in model group were decreased(p<0.01),but mice treated with EE and FA only could increase the ratio of GR/MR in hippocampus(p<0.05)but not in prefrontal cortex.The protein expression of GR,MR and BDNF in the prefrontal cortex were significantly improved after model mice treated with EE or FA(p<0.05 or p<0.01),and the protein expression levels of 11β-HSD1 in the prefrontal cortex were significantly reduced after model mice treated with EE or FA(p<0.05 or p<0.01).Conclusion: Our research results shown the enriched environment and folic acid could ameliorate the anxiety and depression-like behaviors and cognitive dysfunction in model group mice.The mechanism might be due to partly change the DNA methylation status of GR,MR,BDNF and 11β-HSD1 gene,leading to changes in mRNA levels and protein expression levels,causing changes in HPA axis function which mitigated anxiety and depression-like behaviors and cognitive dysfunction in mice.In the other hand,5-mC on behalf of genome-wide methylation status,5-hmC representing the whole genome demethylation state.Our results also suggested that an enriched environment and folic acid could adjust the DNA methylation and DNA demethylation of dynamic balance.However,the effects of enriched environment were better than folic acid,which may indicate that an enriched environment has other mechanisms besides DNA methylation. |
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