| Objective To observe the effects of folic acid on AD cell model induced by Aβ1-40 in vitro.To explore the mechanisms and confirm the connection among the folic acid,DNA methylation and AD.To provide science basis for researching the therapeutic and preventive effects of folic acid on AD.Methods The neurons isolated from the hippocampus of infant rats were cultured in serum-free medium in vitro and then induced to AD cell model by Aβ1-40.The neurons were stained by antibodies against β-tubulin-III and divided into five groups:control group of normal cells(10 μmol/L folic acid,Control),AD group of deficient folic acid(0 μmol/L folic acid,Aβ+FA-D),AD group of normal folic acid(10 μmol/L folic acid,Aβ+FA-N),AD group of low folic acid(20 μmol/L folic acid,Aβ+FA-L),and AD group of high folic acid(40 μmol/L folic acid,Aβ+FA-H).After exposed to Aβ1-40 for 48h,cells were exposed to folic acid for other 48h on the 8th day.MTT was used to detect the activity of AD cell model.LDH Assay kit was used to detect the activity of LDH.FCM was used to detect cell apoptosis rate.The intracellular concentrations of SAM and SAH were examined by HPLC.DNMTs activities were measured by Active Motif’s DNA Methyltransferase Activity/Inhibition Assay kit.Global DNA methylation levels were detected by the methyflashTM methylated DNA Quantification kit.The mRNA expression levels of APP,PS1,PS2 and DNMTs were determined by RT-PCR.The protein expression levels of APP,PS1,PS2 and DNMTs were detected by Western Blot.Results After 7d cultured in serum-free medium,neuron nucleus were clear and neurons were adherent growth in the culture medium.Immunfluorescence staining of cells showed positive result by β-tubulin-III.After AD cell model was exposed to folic acid in vitro,compared with Aβ+FA-N group,neurons were in poor state in AP+FA-D group whereas in normal state in Aβ+FA-L group and Aβ+FA-H group.Compared with A(3+FA-N group,cell activity decreased in Aβ+FA-D group but increased in Aβ+FA-L group and Aβ+FA-H group(P<0.05).Compared with Aβ+FA-N group,LDH activity increased in Aβ+FA-D group but decreased in Aβ+FA-L group and Aβ+FA-H group(P<0.05).Compared with Aβ+FA-N group,cell apoptosis rate increased in Aβ+FA-D group but decreased in Aβ+FA-L group.and FA-H group(P<0.05).Compared with AP+FA-N group,the concentrations of SAH increased and SAM/SAH ratio decreased in Aβ+FA-D group,but the concentrations of SAM increased and SAM/SAH ratio increased in Aβ+FA-L group and Aβ+FA-H group(P<0.05).Compared with Aβ+FA-N group,expressions of mRNA of DNMT1,DNMT3a.and DNMT3b were significantly decreased in AP+FA-D group but significantly increased in Aβ+FA-L group and Aβ+FA-H group(P<0.05).Compared with Aβ+FA-N group,expressions of DNMT3a and DNMT3b were significantly decreased in Aβ+FA-D group,whereas expression of DNMT3b was decreased in Aβ+FA-L group and Aβ+FA-H group(P<0.05).The activities of DNMTs suggested that,compared with Aβ+FA-N group,DNMTs activities were significantly enhanced in Aβ+FA-L group and Aβ+FA-H group(P<0.05).Global methylation results revealed,compared with Aβ+FA-N group,a lowly significant decrease in Aβ+FA-D group and a highly significant increase in AP+FA-L group and Aβ+FA-H group(P<0.05).Compared to Aβ+FA-N group,expressions of mRNA of APP,PS1 and PS2 were significantly increased in Aβ+FA-D group but significantly decreased in AP+FA-L group and Aβ+FA-H group(P<0.05).Compared with Aβ+FA-N group,expressions of APP and PS1 were significantly increased in AP+FA-D group,but expression of PS1 was significantly decreased in Aβ+FA-L group and expression of APP was significantly decreased in Aβ+FA-H group(P<0.05).Conclution Folic acid could decrease the expression of related genes of AD and has protective effect on moderate injured neurons induced by Aβ1-40.Folic acid can enhance DNMTs activities by increasing expressions of DNMT1,DNMT3 a and DNMT3b.Neuroprotective effect of folic acid may relate to its ability for providing methyl groups,which can increase expressions of global methylation of neurons. |
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