Human monocytic leukemia THP-1 cells (West China School of Preclinical and Forensic Medicine, Sichuan University, Sichuan, China) were cultured in RPMI 1640 medium supplemented with 15% (v/v) heat-inactivated fetal bovine serum (GE Healthcare Life Sciences, Logan, UT, USA) in a humidified atmosphere of 5% CO₂ at 37°C. The monocytes were initially differentiated into macrophages by the addition of 500 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) for 48 h, following which the macrophages were incubated (37°C, 5% CO₂) in medium with different concentrations of Hcy and its antagonist for 24 h. In the present study, the cells were divided into five groups: Cells (2×10⁶) were exposed to different concentrations of Hcy (50, 100, 200 and 500 µM), as the Hcy group, others were incubated with Hcy (100 µM) and folic acid (FA; 30 µM) and vitamin B₁₂ (VB₁₂; 30 µM), as the treatment group, and the remainder were treated without Hcy, as a control group. The cells in all groups were cultured for another 24 h, and all cells were used for subsequent analysis.

The human FABP4 gene was amplified from the cDNA of THP-1 macrophages. The human FABP4 gene and the pcDNA3.1-EGFP vector were double-digested using EcoRI and HandIII restriction endonucleases (Takara Bio Inc., Otsu, Japan), and the FABP4 gene fragment was then inserted into the pcDNA3.1-EGFP cloning vector to construct the recombinant plasmid, pcDNA3.1-EGFP/FABP4. The recombinant pcDNA3.1-EGFP/FABP4 plasmid was identified using restriction endonuclease digestion analysis and DNA sequencing. According to the manufacturer's protocol, the THP-1 macrophages grown in 6-well plates were transfected with the recombinant plasmid (pcDNA3.1-EGFP/FABP4) and empty plasmid (pcDNA3.1-EGFP), mediated by liposome reagent (Lipofectamine 2000; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) as the recombinant plasmid group and the empty plasmid control group, respectively. The untransfeced group was set as the normal control group. The levels of green fluorescence in the cells were observed using fluorescence microscopy, and the expression levels of FABP4 were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analyses.

Total RNA was extracted from the cultured macrophages using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), following which the RNA was reverse transcribed into cDNA using a RevertAid first strand cDNA synthesis kit (MBI Fermentas, Inc., Vilnius, Lithuania), according to the manufacturer's protocol. The cDNAs (2 µl) were amplified using a SYBR Green PCR kit (MBI Fermentas, Inc.). Primer Premier 5 software (Premier Biosoft, Palo Alto, CA, USA) was used to design the primers. The primer nucleotide sequences of FABP4 (NM_001442.2; https://www.ncbi.nlm.nih.gov/nuccore/NM_001442.2) were as follows: Forward 5′-TACTGAGATTTCCTTCATACTGGGC-3′ and reverse 5′-GCTCTCTCATAAACTCTCGTGGAAG-3′, with a length of 372 bp. The gene expression of FABP4 was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse 5′-AGGGGCCATCCACAGTCTTC-3′. The RT-qPCR reaction was performed using an FTC-3000 real-time PCR detection system (Funglyn, Toronto, Canada), according to the manufacturer's protocol, as follows: 94°C for 10 min, 50 cycles at 94°C for 15 sec, at the annealing temperature of 53°C for 30 sec and 72°C for 30 sec. The RNA level of the gene was acquired from the value of the quantification cycle (Cq) of the RT-qPCR associated with that of GAPDH using the following formula: ΔCq, ΔCq = Cq_GAPDH − Cq_gene (15). The final results were expressed as N-fold differences in the target gene expression, relative to the calibrator, termed 'N_target', which was determined as follows: N_target = 2^{ΔCqsample − ΔCqcalibrator}, where the ΔCq values of the calibrator and sample were determined by subtracting the Cq value of the target gene from the Cq value of GAPDH, as previously described (11).

The cultured macrophages were harvested by scraping with a plastic scraper and washing with phosphate-buffered saline (PBS). The protein was extracted using a protein extraction kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), and protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Nanjing KeyGen Biotech Co., Ltd.). Equal quantities of protein (80 µg) and a known molecular weight marker were loaded onto 12% sodium dodecyl sulfate-polyacrylamide gels (Nanjing KeyGen Biotech Co., Ltd.) and were transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Boston, MA, USA) by electrophoresis at 300 mA for 50 min at 4°C. The membrane was then blocked in 10 ml 5% skimmed milk for 2 h at room temperature with gentle agitation on a platform shaker. The blocked membranes was respectively incubated with a rabbit monoclonal anti-FABP4 antibody (cat. no. MABS172; 1:400 dilution; EMD Millipore) and β-actin antibody (cat. no. TA-09; 1:2,000 dilution; ZSGB-BIQ, Beijing, China) in 10 ml primary antibody dilution buffer overnight at 4°C. The membrane was then washed three times for 10 min/wash with PBS-0.1% Tween-20 (PBST) and incubated with secondary antibody (cat. no. ab6721; goat anti-rabbit horseradish peroxidase-IgG; Abcam, Cambridge, MA, USA) in PBS at 1:2,000 dilution for 2 h at room temperature. Following being washed three times with PBST, the FABP4 on the membrane was incubated with enhanced chemiluminescence (ECL; Elabscience Biotechnology Co., Ltd., Wuhan, China) for 1 min at room temperature, following which the excess ECL solution was drained, and the membrane was wrapped in a plastic wrap and exposed to X-ray film (Kodak, Shanghai, China). The densities of the bands were quantified and the relative values were normalized to that of β-actin using the following formula: Associated value = experimental densitometry value / β-actin value).

Genomic DNA from the macrophages was isolated using a Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA) and modified using an EZ DNA Methylation-Gold™ kit (Zymo Research Corp., Orange, CA, USA), following which it was used as a template for ntPCR. Following bisulfite modification of the genomic DNA, unmethylated cytosine residues were converted into uracil. The FABP4 DNA methylation primers for MSP amplification were designed using the MethPrimer bioinformatics program (http://www.urogene.org/methprimer/index.html). The inner and outer primers were designed and synthesized based on the DNA sequences of FABP4 (Table I).

To reduce mispriming and to increase efficiency, ntMSP was used for amplification, which comprised a two-step PCR amplification procedure following the standard sodium bisulfite DNA modification. In accordance with the specification requirements of DNA remodified sodium, 4 µl of modified DNA was obtained, 1 µl upstream and 1 µl downstream of each primer, using Go Taq Colorless Master mix (×2; 12.5 µl; Promega), with nuclease-free water added to 25 µl. The samples were subjected to the following steps in a thermal cycler: 94°C for 5 min, 94°C for 30 sec, 74°C for 30 sec and 72°C for 1 min, for 30 cycles, decreasing 0.5°C every cycle, 94°C for 30 sec, for 59°C 30 sec and 72°C for 1 min, 20 cycles, and 72°C for 7 min. The PCR products were separated by electrophoresis on 2% agarose gel. The bands were visualized under ultraviolet illumination and calculated using the following formula: Methylation (%) = methylation / (methylation + unmethylation) × 100%. Each sample was examined using ntMSP analysis three times.

The macrophages were scraped off the culture plate using a rubber scraper and washed in PBS three times, following which they were re-suspended in 0.5 ml PBS (pH 7.4) and lysed by ultrasound for 1 min. The TC was determined, according to the manufacturer's protocol of the Total cholesterol kit-CHO (Beijing BHKT Clinical Reagent Co., Ltd, Beijing, China). TC concentrations were expressed as mM/l.

Experiments were performed at least three times and the results are expressed as the mean ± standard error of the mean. The data were analyzed using one-way analysis of variance, and additional analysis was performed using the Student-Newman-Keuls test for multiple comparisons within different groups. GraphPad Prism 5.01 software was used for analysis (version 5.01, GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The THP-1 monocytes were observed to grow in suspension and not adhere to the plastic surfaces of the culture flasks (Fig. 1A). For the induction of terminal differentiation into macrophages, the THP-1 cells were cultured in the presence of 500 nM PMA for 2 days, and then co-cultured with 50, 100, 200 and 500 mM Hcy or 100 mM Hcy+VB₁₂+FA for 24 h. Following 72 h of culture, the cells had adhered and spread across the bottom of the dish, and had the morphological characteristics of macrophages (Fig. 1B).

To investigate whether the regulation of FABP4 occurred in the context of Hcy, the present study examined the mRNA and protein expression levels of FABP4 using RT-qPCR and western blot analyses in macrophages exposed to Hcy for 24 h. The data demonstrated that the expression of FABP4 was substantially upregulated following incubation with Hcy for 24 h at various concentrations (between 50 and 500 µM), and the mean mRNA expression levels of FABP4 in the experimental groups (50, 100, 200 and 500 mM Hcy, and 100+VB₁₂+FA) were 10.84, 11.46, 5.89, 3.45 and 1.41 times higher, compared with that in the control group, respectively (Fig. 2A). The highest level of expression was observed at the concentration of 100 mM Hcy (P<0.01). In addition, the relative mRNA expression level of FABP4 in the 100+VB₁₂+FA group decreased significantly (87.69%) compared with the 100 µM Hcy group (P<0.01). The effects of Hcy on the protein levels of FABP4 (Fig. 2B) were measured using western blot analysis, which showed similar results to the mRNA levels. The protein expression levels of FABP4 in the 100 and 200 µM Hcy groups were 3.27 and 2.64 times higher, compared with that in the control group (P<0.05), respectively. FA and VB₁₂ also affected the protein expression of FABP4 significantly, and the relative protein expression of FABP4 in the 100+VB₁₂+FA group was significantly decreased by 52.52%, compared with that in the 100 µM Hcy group (P<0.05). However, as the concentrations of Hcy increased, no dose-dependent increase in the mRNA or protein levels of FABP4 were observed. These results indicated that Hcy affected the expression of FABP4 at the transcriptional and translational levels.

DNA methylation patterns have been considered a useful molecular marker for AS, and Hcy can have a global effect on DNA methylation and activate various target genes (16). To determine the effects of Hcy on FABP4 DNA methylation and investigate DNA methylation in the promoter region of FABP4, the THP-1 monocyte-derived macrophages in the present study were treated with Hcy at various concentrations for 24 h, and the DNA methylation status of the FABP4 promoter region of the THP-1 macrophages was determined via ntMSP. As shown in Fig. 3, an increase in the concentration of Hcy between 50 and 100 mM, and a further increase to 500 mM, led to statistically significant differences in the levels of FABP4 promoter region DNA methylation, compared with the control group. The methylation changes in the 100 mM group were the most marked. It was also found that, in the experimental groups (50, 100, 200 and 500 mM Hcy, and 100+VB₁₂+FA), the levels of FABP4 DNA methylation decreased significantly, by 18.84, 43.38, 23.65, 16.50 and 25.28%, respectively. The level of FABP4 DNA methylation in the 100+VB₁₂+FA group increased significantly (31.97%), compared with the 100 mM Hcy group (P<0.01; Fig. 3). These results indicated that Hcy decreased the level of FABP4 promoter region DNA methylation.

The present study also assessed whether Hcy accelerated cholesterol accumulation in the macrophages. The human THP-1 macrophages were treated with increasing concentrations of Hcy, and intracellular TC accumulation was assessed using a Total Cholesterol kit. Hcy significantly increased intracellular TC accumulation, compared with the control group, and the highest level was observed at a concentration of 100 µM Hcy (P<0.01). However, the Hcy-induced increase in intracellular TC accumulation did not occur in a dose-dependent manner (Fig. 4).

The recombinant vector encoding FABP4 was constructed, and it was confirmed that the human FABP4 fragment had been successfully inserted into the pcDNA3.1-EGFP fluorescent eukaryotic expression vector. The FABP4 PCR products were 412 bp on the 1.5% agarose gel (Fig. 5A). The 412 bp fragment encoding human FABP4 was inserted into the pcDNA3.1-EGFP plasmid between the EcoRI and HindIII sites. The successfully constructed recombinant plasmid, pcDNA3.1-EGFP/FABP4, was cut into two fragments using EcoRI and HindIII (Fig. 5B). In addition, the results of the DNA sequencing were identical to the FABP4 sequence reported on GenBank confirming the results.

The recombinant expression plasmid containing the FABP4 gene was successfully transfected into the THP-1 macrophages, and the effective mRNA and protein expression of FABP4 were also confirmed using RT-qPCR and western blot analyses, respectively. The data demonstrated that the expression of FABP4 was significantly upregulated in the recombinant plasmid group, with the mean mRNA expression of FABP4 in the recombinant plasmid group being 3.90 and 3.85 times higher than those in the normal control group and empty plasmid control group, respectively (P<0.01; Fig. 6A). The protein levels of FABP4 were measured using western blot analysis, which showed similar results to those observed for mRNA expression (Fig. 6B). The protein expression of FABP4 in the recombinant plasmid group was higher, compared with the normal control group and empty plasmid control group, (69.79 and 63.36%, respectively; P<0.01). Therefore, the pcDNA3.1-EGFP/FABP4 recombinant fluorescent eukaryotic expression vector was successfully constructed and effectively expressed in the THP-1 macrophages, which provided an experimental basis for further experiments.

The present study subsequently investigated whether FABP4 accelerated cholesterol accumulation in the macrophages. The human THP-1 macrophages were transfected with the pcDNA3.1-EGFP plasmid and pcDNA3.1-EGFP/FABP4 recombinant plasmid, respectively, and TC levels were examined. As shown in Fig. 7, intracellular TC accumulation in the recombinant plasmid group was significantly higher, compared with that in the normal control group and empty plasmid control group (P<0.01). These results indicated that the overexpression of FABP4 significantly increased intracellular TC accumulation. These observations were consistent with a previous report which suggested a critical role for FABP4 in macrophage lipid accumulation (17).