| Objective: Hyperhomocysteinemia （HHcy） is an independent risk factor for thedevelopment of atherosclerosis （AS）. Fatty acid-binding proteins4（FABP4） is involved intrafficking intracellular fatty acids and other lipid signals by interaction with functional targets.However, the underlying mechanism of AS and FABP4in HHcy has not been elucidated. Ourprevious studies demonstrated an important interaction between DNA methylation andhomocysteine （Hcy） in vascular smooth muscle cells and macrophages. The objective of thepresent study was to investigate the effects of hyperhomocysteinemia （HHcy） onatherosclerotic plaque of ApoE-/-mice and interventions of folic acid and vitamin B12（VB12）.Investigate the effect of HHcy on FABP4in vivo and the underlying mechanism ofHcy-induced DNA methylation in ApoE-/- mice. Therefore, to explore these questions inadvance will help clarify the mechanism of Hcy in AS occurred, and it will provide a newperspective for the prevention and treatment of AS.Medthods: Thirty six5-week old C57BL/6J ApoE-/-mice were randomly divided intothree groups, including the Chol group, the Methchol group and the Methchol-FV group, with12mice in each group. Besides, another12normal C57BL/6J mice were served as controlgroup. Serum homocysteine （Hcy） and lipid changes were detected14-week later, and thechanges of plaque size, thickness of intimal and medial were measured by HE staining. Then,total RNA was extracted with Trizol reagent, using real-time fluorescent quantitative PCR（RT-PCR） to detect FABP4mRNA expression related in ApoE-/-mice. Immunofluorescence staining was used to detect the expression of FABP4proteins. In addition, DNA was extractedfrom the the aorta of ApoE-/-mice, using nested-touchdown methylation-specific PCR（ntMS-PCR） to detect the changes of FABP4DNA methylation. Besides, the changes ofDNMT1mRNA were detected by RT-PCR and SAM and SAH were determined by using themethod of HPLC.Results: The serum Hcy levels of the Methchol group were significantly higher thanthe control group and the Methchol-FV group （P<0.01）. The content of LDL, TG and CHOLof the model group increased by2.2,7.6and7.2times than that of the control group, and thecontent of HDL decreased63%（P<0.01）. Atherosclerotic fatty plaque can be seen in thehyperlipidemic, model and intervention group. Aortic changes were attenuated in treatmentgroup when compared to those in the model group. Serum Hcy is more closely related toatherosclerosis （AS）（r=0.4898, P<0.001）.The results of RT-PCR showed that FABP4mRNA expression increased, the Methchol groupwas the most significant group （P<0.01）.The expression results of FABP4proteins detected byimmunofluorescence staining were in accordance with the expression of mRNA.The results of FABP4genes DNA methylation detected by ntMS-PCR showed that: comparedwith the control group, the levels of FABP4gene DNA methylation in the Chol,Methchol andMethchol-FV group decreased by23％,44％and19％respectively, whereas FABP4geneDNA methylation increased1.43times compared the Methchol group with the Methcholgroup, the difference of Methchol group was the most significant（P<0.05）.The results of HPLC showed that compared with the control group, the levels of SAM in theChol and Methchol group increased by2.02and2.96respectively, and the levels of SAH inthe Chol and Methchol group increased by1.27and1.69respectively, while SAM/SAH in theChol and Methchol group increased by1.59and1.73respectively, whereas the levles ofSAM,SAH and SAM/SAH decreased55%,38%and26%respectively compared the Methchol group with the Methchol group, the difference of Methchol group was the mostsignificant（P<0.01）.The results of RT-PCR showed that DNMT1mRNA expression decreased, the Methcholgroup was the most significant group （P<0.05）.Conclusion:1. HHcy can induce the formation of AS in ApoE-/-mice.2. HHcy can promote the formation of AS through affecting the expression of FABP4mRNA and protein.3. HHcy can promote the formation of AS through FABP4promoter region demethylation,and the demethylation can promote the expression of FABP4mRNA and proteinincreasing.4. Earlier folic acid and VB12interference can effectively prevent the vascular injury byHHcy. |
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