FABP4Mediates Homocysteine-induced Cholesterol Accumulation In THP-1Monocyte-derived Macrophages And The Potential Epigenetic Mechanism

Objective: To observe the effects of homocysteine (Hcy) and its antagonists folic acid(FA) together with vitamin B12(VB12) on macrophages by detecting the changes of lipidmetabolism-related gene fatty acid binding protein4(FABP4) mRNA and protein expression,the levels of FABP4DNA methylation, the accumulation of total cholesterol (TC), oxidizedlow density lipoprotein (ox-LDL), endoplasmic reticulum stress indicators such asglucose-regulated protein78(GRP78), X-box binding protein1(XBP1) andCCAAT/enhancer-binding protein homologous protein (CHOP). Clarifying the influence ofHcy on FABP4expression and the methylation regulation on the cholesterol accumulation inTHP-1monocyte-derived macrophages. Using recombinant technology on the critical targetFABP4in order to further explore the effects of FABP4on cholesterol accumulation in THP-1macrophages. And Providing a new theoretical basis for early molecular diagnosis andprevention of atherosclerosis (AS) by using FABP4DNA methylation, and also providing anew perspective for the development of laboratory medicine.Methods: THP-1monocytes were cultured in25cm2flasks with4×106, added5mlculture medium which containing500nM phorbol12-myristate13-acetate (PMA) andcultured in37℃5%CO2for48h. When observed under microscope, the cells were adherent,irregular in shape and had extended pseudopodia, which confirmed that THP-1monocyteswere differentiated into macrophages. Then discarded the old culture medium and washedwith Phosphate Buffered Saline (PBS, pH7.4), replaced it with50,100,200,500M Hcyand100M Hcy+folic acid (FA)+vitamin B12(VB12) RPMI-1640medium containing15% fetal calf serum and cultured for24h, set the group without Hcy as the control group, and setpcDNA3.1-EGFP empty plasmid control group and FABP4recombinant plasmid group.Using quantitative PCR (qRT-PCR) to detect FABP4mRNA expression, using Westernblotting to detect the expression of FABP4proteins. Extracting DNA from the macrophagesand using nested-touchdown methylation-specific PCR (ntMS-PCR) to detect the changes ofFABP4DNA methylation. The contents of total cholesterol (TC) in macrophages weredetected by enzymatic kits. Besides, the contents of ox-LDL, GRP78, XBP1, CHOP weretested by ELISA.Results:1. Successfully replicated the THP-1monocyte-derived macrophage model.2. After intervened with Hcy, the results of ELISA showed that the contents of GRP78, XBP1,CHOP and ox-LDL in macrophages increased significantly, but it’s not in a dose-effectrelationship with Hcy concentrations. There were significant differences (P<0.05) comparedthe experimental groups (50,100,200,500M Hcy and100+FA+VB12) with the controlgroup (0M Hcy), and the most obvious was100M Hcy group, which had a significantdifference(P <0.01). Meanwhile, in100+FA+VB12group, the values of GRP78, XBP1,CHOP and ox-LDL in macrophages reverted back to the levels observed in the controlgroup.3. After intervened with Hcy, The results of enzymatic kits showed that the contents of TC inmacrophages increased significantly compared the experimental groups with the controlgroup (P<0.05). Meanwhile, it’s not in a dose-effect relationship with Hcy concentrations,and the most obvious is100M Hcy group with a significant difference (P<0.01) comparedwith the control group. It indicated that Hcy reduced the cholesterol outflow in THP-1macrophages, hence the intracellular cholesterol accumulation in macrophages increasedsignificantly.4. The results of qRT-PCR showed that FABP4mRNA expression increased,100M Hcy group was the most significant group (P<0.01); The expression of FABP4protein detectedby Western blotting were in accordance with the expression of mRNA.5. The results of FABP4gene DNA methylation detected by ntMS-PCR showed that: after theintervention with Hcy, compared50,100,200and500M Hcy groups with the controlgroup (0M Hcy), the levels of FABP4gene DNA methylation decreased by17.97%、30.47%、18.69%and18.63%, the difference of100M Hcy group was the most significant(P<0.01). Whereas FABP4gene DNA methylation increased by34.28%compared the100+FA+VB12with the control group(P<0.01).6. The FABP4recombinant fluorescent eukaryotic expression vector was constructed andtransfected into THP-1monocyte-derived macrophages, then the expression of FABP4mRNA and protein was detected by quantitative RT-PCR and Western blotting. The resultsshowed that the expression of FABP4mRNA and protein in recombinant plasmid groupwas increased significantly compared with control group (P<0.01). It was identified that theFABP4gene fluorescent eukaryotic expression vector was constructed successfully, andsuccessfully transfected into THP-1monocyte-derived macrophages.7. After FABP4recombinant plasmid was transfected into macrophages, the intracellularcholesterol accumulation in macrophages was significantly increased, and there was asignificant difference compared FABP4recombinant plasmid transfection group with thecontrol group (P<0.01). It indicated that FABP4reduced the cholesterol outflow withinmacrophages, thereby increasing TC content significantly.Conclusions: In THP-1monocyte-derived macrophages, Hcy can increase theendoplasmic reticulum stress indicators such as GRP78, XBP1and CHOP, and Hcy canpromote the increasing of TC and ox-LDL. Meanwhile, Hcy can decrease the lipidmetabolism-related gene FABP4DNA methylation levels by causing methionine cycledisorder, thereby increasing the expression of FABP4mRNA and protein and leading to anincreased accumulation of cholesterol in macrophages. So it plays an important role in promoting the occurrence and development of AS.