FABP4-mediated homocysteine-induced cholesterol accumulation in THP-1 monocyte-derived macrophages and the potential epigenetic mechanism
FABP4-mediated homocysteine-induced cholesterol accumulation in THP-1 monocyte-derived macrophages and the potential epigenetic mechanism
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FABP4介导的THP-1单核细胞来源的巨噬细胞中同型半胱氨酸诱导的胆固醇积累及其潜在的表观遗传机制
DOI：
10.3892/mmr.2016.5315
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发表时间：
2016-07-01
影响因子：
3.4
通讯作者：
Shi, Yingkang
中科院分区：
文献类型：
--
作者：
Jiang, Yideng;Ma, Shengchao;Shi, Yingkang
大量的临床和流行病学研究表明，高同型半胱氨酸血症（HHcy）是动脉粥样硬化（AS）的独立危险因素。然而，HHcy促进AS的潜在致病分子机制仍有待充分阐明。脂肪酸结合蛋白4（FABP 4）在巨噬细胞胆固醇运输中具有重要作用。本研究的目的是确定同型半胱氨酸（homocysteine，Hcy）是否通过调节FABP 4而加速AS，进而介导巨噬细胞内胆固醇蓄积。分别将0、50、100、200和500 μ M的Hcy和100 μ M Hcy+30 μ M维生素B-12（VB 12）+ 30 μ M叶酸（FA）加入培养的THP-1单核细胞衍生的巨噬细胞24 h。在巨噬细胞中使用逆转录-定量聚合酶链反应（RT-qPCR）和蛋白质印迹分析来确定FABP 4的水平，FABP 4作为将细胞脂质积累与炎症联系起来的关键因素。本研究采用巢式降落甲基化特异性PCR检测FABP 4启动子区DNA甲基化状态。将FABP 4基因片段克隆到真核表达载体pcDNA3.1-EGFP中，构建重组质粒pcDNA3.1-EGFP/FABP 4。通过脂质体试剂介导，将pcDNA3.1-EGFP/FABP 4表达质粒转染入THP-1单核细胞源性巨噬细胞，然后采用RT-qPCR和western印迹分析检测FABP 4的表达水平。本研究还测定了巨噬细胞中总胆固醇的细胞内蓄积。结果表明，与对照组（0 μ M Hcy）相比，Hcy降低了巨噬细胞FABP 4启动子甲基化水平，但增加了FABP 4 mRNA和蛋白表达水平。然而，没有剂量依赖性的变化，观察到随着同型半胱氨酸浓度的增加。成功构建了荧光真核表达载体pcDNA3.1-EGFP/FABP 4，并在THP-1巨噬细胞中进行了高效表达。结果还表明，FABP 4加速了巨噬细胞中胆固醇的积累。综上所述，本研究的结果表明，Hcy诱导的FABP 4 DNA低甲基化可能参与了FABP 4的过表达，从而诱导巨噬细胞中胆固醇的积累。
Hyperhomocysteinemia (HHcy) is an independent risk factor for the development of atherosclerosis (AS), according to overwhelming number of clinical and epidemiological studies. However, the underlying pathogenic molecular mechanisms by which HHcy promotes AS remain to be fully elucidated. Fatty acid binding protein 4 (FABP4) has been shown to be important in macrophage cholesterol trafficking. The objective of the present study was to determine whether homocysteine (Hcy) accelerates AS through regulating FABP4, and then mediates cholesterol accumulation in macrophages. Hcy concentrations of 0, 50, 100, 200 and 500 mu M, and 100 mu M Hcy+30 mu M vitamin B-12 (VB12)+ 30 mu M folic acid (FA) were respectively added to cultured THP-1 monocyte-derived macrophages for 24 h. The levels of FABP4, which acts as a key factor connecting cellular lipid accumulation to inflammation, were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analyses in the macrophages. The present study used a nested touchdown methylation-specific PCR assay to detect the DNA methylation status of the FABP4 promoter region. In addition, the FABP4 gene fragment was inserted into the cloning vector, pcDNA3.1-EGFP, to construct the recombinant plasmid, pcDNA3.1-EGFP/FABP4, which was identified using restriction endonuclease digestion analysis and DNA sequencing. The pcDNA3.1-EGFP/FABP4 expression plasmid was transfected into THP-1 monocyte-derived macrophages, mediated by liposome reagent, following which the expression levels of FABP4 were detected using RT-qPCR and western blot analyses. The present study also determined the intracellular accumulation of total cholesterol in the macrophages. The results indicated that Hcy decreased the levels of FABP4 promoter methylation, but increased the mRNA and protein expression levels of FABP4 in the macrophages, compared with the control group (0 mu M Hcy). However, no dose-dependent changes were observed with increasing concentrations of Hcy. The recombinant fluorescent eukaryotic expression vector, pcDNA3.1-EGFP/FABP4, was successfully constructed and effectively expressed in the THP-1 macrophages. The results also showed that FABP4 accelerated the accumulation of cholesterol in the macrophages. Taken together, the results of the present study suggested that FABP4 DNA hypomethylation induced by Hcy may be involved in the overexpression of FABP4, thereby inducing cholesterol accumulation in macrophages.