| ObjectiveCardiovascular diseases?CVDs?are chronic disease that can seriously endanger human life.Atherosclerosis?AS?is the principal pathological cause of CVDs.Recently,various studies have analyzed the involvement of epigenetic mechanisms in the development and progression of AS.A dietary deficiency of folic acid,an important component of transmethylation micronutrients in one carbon metabolism,has been linked to endothelial dysfunction and AS.However,the mechanism of the effect of folic acid supplementation in atherosclerotic process remains unclear.In the present study,we hypothesized that folic acid functions through an epigenetic gene silencing mechanism to lower AS-related and oxidative stress related gene expression in ApoE-/-mice and human umbilical vein endothelial cell?HUVEC?.MethodsThis study consists of three parts.Part 1:Determination of ApoE-/-mice model and HUVEC Model of ox-LDL damage.?1?A total of 24 homozygous male ApoE-/-mice on a C57BL/6J background aged 4 weeks were distributed into 4 groups.All mice were fed with specific diets and gave intragastrical administration for 20 weeks.Oil red O staining and HE staining were used to assess pathological changes.?2?HUVECs were exposed to different concentration of ox-LDL for 24 h and then detected of cell viability and MDA level.All the HUVECs in the treatment groups were exposed to the indicated concentrations of folic acid?0–1000 nmol/L?for 48 h.MTS was used to assess cell viability.Meanwhile,observed the changes of cell morphology.Part 2:Effect of folic acid on methylation metabolism.We analyzed serum and intracellular folate concentration by chemiluminescence immunoassay analyzer,plasma and intracellular HCY,SAM and SAH concentration by HPLC,aortic tissue and intracellular DNA methyltransferase activity by Elisa,MCP1 and VEGF promoter methylation by Sequenom Methylation,MCP1 and VEGF expression by RT-PCR and Western Bolt.The folic acid pathway was verified by DNMTs inhibitor in the HUVECs model.Part3:Folic acid improves endothelial function by DNA methylation-oxidative stress pathway.We analyzed VPO1 promoter methylation by Sequenom Methylation,VPO1expression by RT-PCR and Western Bolt,serum ox-LDL level by Elisa,intracellular ROS and apoptosis rate were detected by flow cytometry,aortic tissue and intracellular8-OH-dG concentration by immunofluorescence and Elisa,aortic tissue apoptosis rate detected by immunohistochemical staining.ResultsPart 1:AS models in vivo and in vitro were established.?1?AS animal model has been successfully established by high-fat feeding ApoE-/-mice for 20 weeks.Folic acid reduced atherosclerotic lesion size in ApoE knockout mice?P<0.05?.?2?The growth inhibition ratios for 120?g/mL ox-LDL group were closest to 50%.500-1000 nmol/L folic acid addition could significantly increase the cell viability in ox-LDL-induced HUVECs model?P<0.05?.Part 2:In ApoE-/-mice and HUVECs treated with ox-LDL,folic acid raised the folate concentration,upregulated the plasma Hcy concentration and SAM:SAH ratio?P<0.05?,elevated DNA methyltransferase activity and expression,altered MCP1 and VEGF promoter methylation,inhibited MCP1 and VEGF expression?P<0.05?.Part 3:Folic acid altered VPO1 promoter methylation,and inhibited VPO1 expression in ox-LDL-induced HUVEC model.Folic acid decreased ROS level in HUVEC model?P<0.05?.Folic acid downregulated 8-OH-dG concentration and cell apoptosis rate in ApoE-/-mice and HUVEC model.ConclusionIn conclusion,the underlying folic acid delays atherosclerotic development mechanism appears to operate through increased the methylation potential and DNMT activity,modified DNA methylation,ameliorated MCP1,VEGF and VPO1 levels,reduced the accumulation of ROS,alleviated DNA damage,inhibited apoptosis rate,improved vascular endothelial dysfunction.This study identified the specific gene dna methylation pattern of folic acid delaying AS development,it may provide new insights regarding the use of folic acid as a nutritional adjunct to prevent AS in high-risk individuals. |
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