The Mechanism Of EC-SOD DNA Methylation In Hyperhomocysteinemia-induced Atherosclerosis In ApoE^-/- Mice

Objective To study the efftct of EC-SOD DNA methylation in hyperhomocysteinemia-induced atherosclerosis（AS） in ApoE^-/-mice and to further explore and clear the mechanism inTHP-1monocyte-derived macrophages. Try to find the key target of EC-SOD methylationchanges caused by Hcy and to provide a new theoretical and experimental basis for theprevention and treatment of HHcy-induced AS.Methods1. Thirty six5-week old C57BL/6J ApoE^-/-mice were randomLy divided intothree groups, including the ApoE^-/-group, the HHcy group and the intervention group, with12mice in each group. Besides, another12normal C57BL/6J mice were fed with regular mousediet as the normal control group. After14weeks, the mice in each group were anesthetized（2mL/100g20%ethylcarbamate enterocoelia injection）. The blood was collected byextraction the eyeball and the serum was separated by centrifugation. Serum homocysteine（Hcy） and lipids （total cholesterol （TC）, triglyceride （TG）, high density lipoprotein （HDL） andlow density lipoprotein （LDL）） was detected by ADVIA2400Automatic biochemistryanalyzer. The aorta were made into paraffin sections and stained with haematoxylin and eosin.The changes of T-AOC、MDA、O₂-·、OH·、H₂O₂contents were measured by chromatometry.The mRNA expressions of EC-SOD and DNMT1were detected by real-time PCR. Theprotein expression of EC-SOD was analyzed by immunofluorescence. The methylation levelsof EC-SOD were detected by nested-touchdown methylation-specific PCR（ntMS-PCR）.S-adenosylmethionine （SAM） and S-adenosylhomocysteine （SAH） concentrations wereassayed by high-performance liquid chromatography （HPLC）.2. THP-1monocytes were cultured in the25cm2flasks with4×106. Then added5mL R/MINI1640medium including500nmol/L phorbol myristate acetate （PMA） and cultured at37°C in a5%CO₂atmosphere for48h. When observed the cells were adherent, irregular inshape and had extended pseudopodia under the microscope, which confirmed that THP-1cellswere differentiated into macrophages. To construct the pEGFP-N1-DNMT1recombinantexpression vector and use Lipofectamine2000to transfect macrophages. To observe theexpression of recombinant vector in macrophages and take pictures under a fluorescencemicroscope, and then collect the cells.The mRNA and protein expressions of DNMT1wereanalyzed by real-time PCR and Western blotting. The methylation levels of EC-SOD weredetected by ntMS-PCR.Results1. The levels of serum Hcy and lipids in the HHcy group were significantlyhigher than the normal control group and the ApoE^-/-group （P<0.01）. Slices with the HEstaining under the microscope showed that the aortic intima of the HHcy group visiblethickening and formed obvious atherosclerotic fatty plaque, visible subendothelialaccumulation of foam cells. The H₂O₂content of the HHcy group increased than that of thecontrol group, but the content of T-AOC、inhibiting rate of O₂-· and OH· decreased （P<0.05,P<0.01）. However, the intervention group has the opposite results compared with the HHcygroup.The mRNA and protein levels of EC-SOD were increased in the HHcy group comparedwith other three groups （P<0.05, P<0.01）.The methylation levels of EC-SOD detected byntMS-PCR showed that the HHcy group was lower than that of the control group. After thefolic acid and Vitamin B12intervention, the EC-SOD methylation level increasedsignificantly compared with the HHcy group （P<0.05）. The mRNA expression of DNMT1inthe HHcy group was decreased34.23%and27.53%, compared with the normal control groupand the ApoE^-/-group （P<0.05）. SAM and SAH concentrations determined by HPLC showedthat the HHcy and ApoE^-/-group both increased significantly compared with the normalcontrol group, and there is a significant difference （P<0.01）.2. The eukaryotic expression vector pEGFP-N1-DNMT1was successfully constructed. After the recombinant vector was successfully transfected into macrophages, massive greenfluorescence protein could be observed under the fluorescence microscope. The real-timePCR results showed that DNMT1mRNA expression of the pEGFP-N1-DNMT1group wassignificantly higher than that of the normal control group and the pEGFP-N1group. Theprotein expression of DNMT1detected by western blotting was unanimously with its mRNA.The ntMS-PCR results showed that the EC-SOD methylation level of100μmol/L Hcy groupdecreased15.67%compared with the normal control group. The EC-SOD methylation levelof pEGFP-N1-DNMT1group respectively elevated1.16-And1.38-fold, compared with thenormal control group and100μmol/L Hcy group. The methylation level of pEGFP-N1-DNMT1+100μmol/L Hcy group was lower than that of pEGFP-N1-DNMT1group.Conclusions Through disturbance the methionine circulation, Hcy can induce thechange of gene DNA methylation status, in which the most important enzyme ofmethyltransfer process DNMT1changes and causes the reduction of EC-SOD DNAmethylation level, thus makers the expression of EC-SOD mRNA and protein upregulation.The eukaryotic expression vector pEGFP-N1-DNMT1was successfully constructed andtransfected into macrophages. DNMT1overexpression resulted in the methylation levels ofEC-SOD increase. The key regulatory role of DNMT1in EC-SOD methylation changes wasclear and provide a possible potential target for AS.