Effect Of DNA Hypomethylation On Maternal Vitamin D Deficiency Induced Depression-like Behavior In Offspring

Objective:Depression is a common mental disorder with increasing incidence.Epidemiological studies have found that vitamin D deficiency during pregnancy increases the incidence of depression in offspring,but the mechanism is unclear.This study aims to investigate whether maternal vitamin D deficiency induces depression-like behaviors in adult offspring by reducing cortical DNA methylation.Methods:First,an animal model of Vitamin D deficiency（VDD）was established.Female ICR mice（5 weeks old）were purchased.Female mice were randomly equated into two groups.In the VDD group,mice were given a complete vitamin D deficient diet（0 IU/kg vitamin D₃）while avoiding light exposure.In the control group,mice were fed a standard diet（1000 IU/kg vitamin D₃）.After continuous feeding for 4weeks,they were mated with normal ICR male mice.The pregnant mice were identified as Gestational Day 0（GD0）.This study consists of three experimental parts.In experiment one,to investigate the effects of maternal vitamin D deficiency during pregnancy on depression-like behavior in offspring adult mice.Pregnant mice in the VDD group continued to receive a vitamin D deficiency diet throughout pregnancy,and pregnant mice in the control group continued to receive a standard diet throughout pregnancy.Pregnant mice in the VDD group continued to receive a vitamin D deficiency diet throughout pregnancy,and pregnant mice in the control group continued to receive a standard diet throughout pregnancy.Pregnant mice were dissected in each group on GD18.Serum and cerebral cortex tissues of maternal and fetal mice were collected.The remaining pregnant mice were delivered naturally,and on the first Day after birth（Postnatal Day 1,PND1）,all the female mice were given a standard diet,and the offspring mice were adjusted according to the principle of nest balance（female and body weight）,PND35 male and female mice were divided into cages,and PND70 female mice were prepared.12 male and 12 female mice in each group were randomly selected for a depression-like behavior experiment（sugar preference,tail suspension,forced swimming）.All mice were sacrificed two weeks after completing the behavioral experiment.Cortex samples were collected.In experiment two,the mechanism of vitamin D deficiency leads to depression-like behavior in offspring in adulthood.Folate levels in the serum of mice were measured by chemiluminescence immunoassay.Transcriptomics was used to screen the differential expression of depression-related genes in the cortex.RT-PCR was used to detect the m RNA expression of depression-related genes in the cortex of offspring mice.The content of S-adenosine methionine（SAM）and the whole genome DNA methylation in mice cortex was determined by Liquid chromatography-tandem mass spectrometry（LC/MS-MS）.DNA methylation of Cp G fragments was detected by transcriptome and target region methylation sequencing.In experiment three,to analyze the protective effect of maternal folic acid（FA）supplementation on depression-like behavior in offspring,pregnant mice were divided into control,FA,VDD,and VDD+FA groups.In the FA and VDD+FA groups,pregnant mice were given folic acid（150μg/m L）daily by gavage,and the control group and VDD group were given the same volume of normal saline intragastric administration.On GD18,some pregnant mice were sacrificed in each group.Serum samples and fetal cortical samples were collected.As for the rest of the pregnant mice in each group,they naturally delivered their pups.Only four female and four male pups were retained in each dam.Depressive-like behavioral experiments with sugar preference,tail suspension,and forced swimming tests were performed in adult offspring（PND70,12males and 12 females for each group）.All mice were sacrificed two weeks after completing the behavioral experiment.Cortex samples were collected.Results:The results showed that VDD adult mice had significantly lower sucrose consumption than control mice,with longer breaks in tail suspension and forced swimming experiments.Compared with the VDD group,sucrose consumption of adult mice in the VDD+FA group was increased,and the rest time was significantly reduced in the tail suspension and forced swimming tests.The results suggest that maternal vitamin D deficiency leads to depression-like behavior in adult offspring,and folic acid supplementation alleviates VDD-induced depression-like behavior in adult offspring.Mechanism analysis showed that m RNA expression levels of depression-related genes in the cerebral cortex of offspring adult mice in the VDD group were significantly up-regulated compared with the control group,such as Ache,Mdga1,Gmip,Lgals3bp,Atp2b4,Oxtr,Smad7,Pcsk9,Htr1b,P2rx4,Ezh2,and Cpt1a.For female adult offspring,methylation of Cp G fragments for Ache and Oxtr was reduced in the VDD group.For male adult offspring,methylation of Cp G fragments of Cpt1a and Htr1b was reduced in the VDD group.The serum folic acid concentration and cortical SAM content of the VDD group were decreased.Conclusion:The above results indicate that gestational vitamin D deficiency may cause a reduction of fetal folate content,which influences cortical DNA methylation and altered m RNA expression levels of depression-related genes,thus inducing depression-like behavior in adult offspring.