The human monocytic cells, THP-1 (ATCC, Rockville, MD, USA), were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/l glucose and 1.5 g/l bicarbonate in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. The differentiation of THP-1 monocytes into macrophages was induced by exposure to 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) for 48 h. The differentiated THP-1 macrophages were extensively washed in PBS before being used in the experiments.

The extraction and oxidation of LDL were performed according to a previously performed method (17). For the production of oxidized LDL (ox-LDL), CuSO₄ was added to LDL at a final concentration of 10 μM for 24 h at 37°C and the mixture was then dialyzed for 24 h at 4°C. Finally, the bacteria in the solution were removed through ultrafiltration.

The THP-1-derived macrophages were divided into an ox-LDL (50 mg/l) group and BBR (5, 10 and 20 mg/l; Wuhan Fortuna Chemical Co., Ltd., Wuhan, China) groups. The experiments were performed in serum-free (SF) experimental medium. The BBR groups were co-cultured with BBR for 2 h prior to the addition of ox-LDL, which was added at a concentration of 50 mg/l. These cells were then collected after having been incubated for 24 h.

The transient transfection of siRNA into THP-1-derived macrophages was performed using Lipofectamine RNAi MAX (Sigma-Aldrich). The oligos used for SIRT1-siRNA have been previously described (9). The negative control (NC) group was transfected with a siRNA sequence which had no effect on gene expression. Subsequently, the cells were exposed to BBR (10 mg/l) with or without ox-LDL (50 mg/l) for 12 h.

Compound C (Sigma-Aldrich) is a specific adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) inhibitor. Compound C (10 μM, in DMSO) was added 15 min prior to treatment with BBR. For treatment, the cells were treated only with BBR (10 mg/l) for 24 h or BBR was added to the cultures to a final concentration of 10 mg/l for 2 h prior to the addition of ox-LDL, which was added at a concentration of 50 mg/l. These cells were then collected after 12 h of incubation.

The THP-1-derived macrophages were divided into atorvastatin (20 and 40 μM; Sigma-Aldrich), BBR (10 mg/l) and atorvastatin plus BBR (atorvastatin 20 μM, BBR 10 mg/l) groups. The experiments were performed in SF experimental medium. The treatment groups were co-cultured with the drugs for 2 h and were then treated with ox-LDL (50 mg/l) or lipopolysaccharide (LPS; 10 μM; Sigma-Aldrich) for 12 h.

The formation of foam cells was evaluated using Oil Red O staining. The THP-1-derived macrophages were cultured with ox-LDL for 24 h and then washed 3 times with PBS. Following fixation with 4% formaldehyde for 15 min, the cells were stained with Oil Red O (3 mg/ml in 60% isopropanol; Sigma-Aldrich) for 10 min at 37°C to evaluate the characteristic lipid accumulation in macrophage-derived foam cells. The cells were then rinsed with water, and hematoxylin was introduced to label the cell nuclei. Foam cell formation was observed under a microscope, and Oil Red O staining was assessed by a color density assay using iVision Software. The density of lipid content was evaluated by alcohol extraction after staining. The absorbance at 540 nm was measured using a microplate reader (BioTek Instruments, Winooski, VT, USA).

Cellular total triglyceride content and cholesterol levels were analyzed by HPLC. Briefly, the cells were rinsed in PBS 3 times and lysed by the addition of a 0.9% NaOH solution. Masterol was used as a standard, and the samples were dissolved in 100 μl of isopropanol-acetonitrile (v/v, 20:80), followed by incubation in ultrasound water at room temperature for 5 min. Finally, the samples were placed in the Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara, CA, USA).

Total RNA was extracted using TRIzol (Sigma-Aldrich) reagent and reverse transcribed into cDNA. The cDNA was quantified by SYBR-Green qPCR using the SYBR Premix Ex Taq™ II kit (Takara Bio Inc., Otsu, Japan). The reaction conditions followed the instructions provided by the manufacturers. Gene-specific primers for monocyte chemotactic protein-1 (MCP-1), SIRT1, PPAR-γ and β-actin (Table I) were used in the reactions.

Protein expression was evaluated by western blot analysis. Specific antibodies for phospho-AMPKα (Thr172), AMPK, SIRT1, PPAR-γ and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA) and Sigma-Aldrich. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (20 μg) from each lysate were subjected to SDS-PAGE. The proteins were transferred onto nitrocellulose membranes at 80 V for 1 h and blocked for 4 h in 5% skim milk, then incubated overnight at 4°C with a 1:500 dilution of primary antibody (produced in rabbit and mouse) followed by incubation with the corresponding secondary antibody (goat anti-rabbit or anti-mouse IgG). The enhanced chemiluminescence system (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) was used for detection. Filters were subsequently exposed to Kodak BioMax light-1films (Eastman Kodak, Rochester, NY, USA) and the intensity of the western blot signals was quantified by densitometry.

To evaluate the levels of MCP-1 produced, the THP-1-derived macrophages were pre-treated with BBR or atorvastatin, or a combination of both for 2 h then treated with LPS for 12 h. Supernatants from the treated cells were collected and analyzed for MCP-1 using a sandwich ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Data are presented as the means ± standard deviation (SD, n=6). All data were evaluated using SPSS 11.0 software. Groups were compared using analysis of variance (ANOVA) followed by the Student’s t-test. P-values <0.05 or <0.01 were considered to indicate statistically significant differences.

To investigate the effects of BBR on ox-LDL-induced foam cell formation, the THP-1-derived macrophages were treated with ox-LDL in the presence or absence of BBR for 24 h. BBR effectively suppressed ox-LDL-induced foam cell formation (Fig. 1A). The increase in lipid and cholesterol accumulation was significantly attenuated by treatment with BBR in a dose-dependent manner (Fig. 1B and C), which suggests that BBR abrogates the formation of foam cells by regulating lipid accumulation.

SIRT1 can reduce the accumulation of fatty acids by repressing PPAR-γ (18). In this study, to explore the molecular mechanisms involved in the lipid lowering effects of BBR, the THP-1-derived macrophages were treated with ox-LDL in the presence or absence of BBR for 24 h and the effects of BBR on the expression of SIRT1 and PPAR-γ were then examined. Treatment with BBR increased the expression of SIRT1 and decreased the expression of PPAR-γ at both the protein and mRNA level in the macrophages in a dose-dependent manner (Fig. 2).

To further investigate the role of SIRT1 in the lipid-lowering effects of BBR, the THP-1-derived macrophages were transfected with SIRT1 siRNA in the presence of BBR. Pre-treatment of the macrophages with SIRT1 siRNA diminished the effects of BBR on the expression of SIRT1 and PPAR-γ at both the protein and mRNA level (Fig. 3A and B). Additionally, pre-transfection with SIRT1 siRNA abolished the BBR-mediated suppression of oxLDL-induced lipid accumulation (Fig. 3C and D), suggesting that the induction of SIRT1 is required for BBR-mediated protection against foam cell formation.

The activation of AMPK has been suggested to play an important role in SIRT1 gene expression and to switch off a number of processes that consume ATP, such as fatty acid, protein and cholesterol synthesis (19–22). In this study, to determine whether AMPK is involved in the BBR-induced upregulation of SIRT1, we examined the expression of AMPK in response to BBR. The phosphorylated isoform is the active AMPK form; thus, we determined the phosphorylated AMPK/total AMPK protein ratio. BBR significantly increased the phosphorylation of AMPK (Fig. 4A) suggesting that AMPK may contribute to the enhancing effects of BBR on SIRT1 expression in macrophages.

To verify this hypothesis, the THP-1-derived macrophages were pre-treated with an AMPK inhibitor (compound C). Pre-treatment of the macrophages with compound C diminished the BBR-induced expression of SIRT1 at both the protein and mRNA level (Fig. 4B). In addition, the suppression of lipid accumulation induced by BBR was reversed when the macrophages were pre-treated with compound C (Fig. 4C and D).

MCP-1 is a potent chemoattractant for monocytes and plays pivotal roles in the initiation and development of atherosclerosis by promoting monocyte infiltration to lesion-prone areas and penetration between endothelial cells into the inner arterial space (23,24). BBR has been used as an anti-inflammatory agent. It has been reported that BBR inhibits the expression of MCP-1 in macrophages (25). Atorvastatin is a traditional anti-atherosclerotic drug. The anti-atherosclerotic effects of atorvastatin may be partly achieved by inhibiting the secretion of MCP-1 and the expression of SIRT1 in foam cells (26,27).

In the present study, we investigated the anti-atherosclerotic effects of a combination of atorvastatin and BBR. As shown in Fig. 5, atorvastatin and BBR both suppressed the LPS-induced expression and secretion of MCP-1, as well as lipid accumulation. In addition, both atorvastatin and BBR upregulated the expression of SIRT1. However, the combination of atorvastatin and BBR was more effective than using atorvastatin alone. These data suggest a strong synergistic benefit of combination therapy with BBR and atorvastatin for preventing atherosclerotic processes.