Effects Of Folic Acid On The APP/PS1 Mouse Model By DNA Methylation

ObjectiveTo observe the effect of folic acid on the APP/PS1 mouse model and explore the underlying mechanism.To provide science basis for researching the therapeutic and preventive effects of folic acid on neurodegenerative diseases.MethodsThe 6-month-old male APP/PS1 mice were randomly assigned to four groups.The groups were:folate-deficient diet plus daily gavage with water(AD+FD),control diet plus daily gavage w:ith water(AD Control),control diet plus daily gavage with 120μg/kg folic acid(AD+120μg/kg bw·d),control diet plus daily gavage with 600μg/kg folic acid(AD+600 μg/kg bw·d).Wild type mice(C57BL/6J)were fed the control and gavaged daily with water(WT Control).The folate-deficient diet(containing folic acid 0.2 mg/kg diet)and the control diet(folic acid 2.1 mg/kg diet)were purchased from TestDiet(St.Louis,America).Weighed mouse body once a week.During 60 days intervention,adjusted intragastic volume according to the body weight.Serum folate concentration was measured by IMMULITE folic acid chemiluminescent immunoassay kit.The plasma concentrations of Hey,SAM and SAH were examined by HPLC method.The expression levels of APP,PS1 and DNMTs in brains were quantified by real-time PCR.The protein expression levels of DNMTs in brains were detected by Western blot.DNMTs activity in brains was measured by Active Motifs DNA Methyltransferase Activity/Inhibition Assay kit.Methylation levels of APP and PS1 gene promoter in brains were determined by MeDIP-qPCR.The expressions of β-amytoid protein and PS1 protein in the brain tissue were measured by immunohistochemistry(IHC).ResultsAfter 60d folic acid intervention,the result of chemical immunoluminometric showed that before the intervention,serum folate concentration in each group was at the same level.1 week after intervention,serum folate levels were changed in 5 groups.AD+FD group serum folate concentration was lower than other groups.serum folate levels in AD+120μg/kg bw.d group and AD+600μg/kg bw·d group were higher than that in AD Control group(P<0.05).Serum folate level was maintained three different levels between five groups(P<0.05).Compared with AD Control group,the concentration of SAM in plasma increased,the concentration of SAH and Hcy in plasma decreased,and SAM/SAH ratio increased in AD+120μg/kg bw·d group and AD+600μg/kg bw·d group(P<0.05).The concentration of SAM,SAH,Hey and SAM/SAH ratio in plasma in AD+FD groups was lower than that in AD Control group(P<0.05).However,the concentration of SAM,SAH,Hcy in plasma did not differ between the AD+120μg/kg bw-d group and AD+600μg/kg bw·d group.The result of RT-PCR and western blot showed that,compared with AD Control group,the expressions of DNMTs gene and protein in brains increased in both AD+120μg/kg bw·d group and AD+600μg/kg bw·d group(P<0.05).DNMT3b gene expression in AD+600μg/kg bw·d group significantly increased(P<0.05).The protein expressions of DNMT3a,DNMT3b in AD+600μg/kg bw-d group significantly increased(P<0.05).Only the gene expression of DNMT3b in brains in AD+FD groups was lower than that in AD Control group(P<0.05).However,the gene expression and protein expression of DNMTs did not differ between AD+120μg/kg bw-d group and AD+600μg/kg bw-d group.The activities of DNMTs suggested that,compared to AD Control group,DNMTs activities were significantly enhanced in AD+120μg/kg bw-d group and AD+600μg/kg bw·d group(P<0.05),while those were inhibited in AD+FD group(P<0.05).However,the DNMTs activities in brains did not differ between the AD+120μg/kg bw·d group and AD+600μg/kg bw·d group.As we could conclude from the MeD1P-qPCR result that,compared with AD Control group,the APP and PS1 promoter revealed a highly significant hypermethylation in AD+120μg/kg bw·d group and AD+60μg/kg bw·d group(P<0.05),while the level of methylation was reduced in AD+FD group(P<0.05).However,the methylation levels of APP and PS1 promoter in brains did not differ between the AD+120μg/kg bw-d group and AD+600μg/kg bw·d group.Compared with AD Control group,gene expressions of APP and PS1 in brains were significantly reduced in AD+120μg/kg bw·d group and AD+600μg/kg bw·d group(P<0.05),the expression of APP and PS1 in brains were increased in AD+FD group(P<0.05).However,the expression of APP and PS1 in brains did not differ between the AD+120μLg/kg bw·d group and AD+600μg/kg bw·d group.Immunohistochemistry results revealed a highly significant decreased of Aβ and PS1 protein expressions in AD+120μg/kg bw·d group and AD+600μg/kg bw·d group(P<0.05)in brains,and the Aβ and PS1 protein expressions level of AD+FD group was higher than that in AD Control group in brains(P<0.05).However,the Aβ and PS1 protein expressions in brains did not differ between the AD+120μgg/kg bw·d group and AD+600μg/kg bw·d group.ConclusionThe experimental results show that folic acid supplementation can reduce brain Aβ deposition.Folic acid deficiency can promote the brain tissue Aβ deposition.The mechanism may be that folic acid has changed the DNA methylation level by alters SAM and SAH concentrations which are critical metabolites involved in DNA methylation pathway.At the same time,folic acid improves the methylation potential and the gene expression and protein levels of DNMTs,it also increases the DNMTs activity and promotes the occurrence of AD-related gene methylation reaction.The increasing level in AD-related genes promoter moderated by folic acid inhibit those genes expression and Aβ production.