A total of 30 9-week-old male LDLR^−/− knockout mice with a C57BL/6 background (Body weight, 24.5±0.37 g) were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China) and housed in ventilated cages maintained at 22±2°C, 55±5% relative humidity with a 12 h light/dark cycle. Food and water were administered ad libitum. Mice were randomly divided into three groups (n=10) and fed with the following; chow diet, western-type diet (Research Diets D12108C formula containing 4.5 kcal/g, 40% of energy from fat and 1.25% cholesterol; Research Diets, Inc., New Brunswick, NJ, USA) or western-type diet supplemented with luteolin (>98% purity; 100 mg/kg diet; Sigma Aldrich; Merck KGaA, Darmstadt, Germany) for 14 weeks. All animal experimental procedure protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Capital Medical University (Beijing, China). At the end of the treatment period, mice were sacrificed and the plasma and tissues were collected and immediately frozen.

Whole abdominal aortas were collected, fixed with 10% formalin for 24 h and stained with oil red O for 2.5 h at room temperature (25°C) to detect the lipid deposition in lesions. Images of the entire aortic intimal surface were captured using a digital camera and digitally scanned (Nikon Corporation; Tokyo, Japan). Oil red O-positive stained lesions were identified by assessing staining intensity with Image-Pro Plus Version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Quantification of the atherosclerotic lesion area was expressed as a positive area percentage.

After 14 weeks, mice were euthanized with CO₂ asphyxiation in a 7.07-l chamber with 1.5 l/min flow rate and a final concentration of 100% CO₂. To confirm sacrifice was successful, mice remained in the chamber filled with 100% CO₂ for an additional time of ~3 min. Subsequently, their blood were harvested. To obtain plasma, blood was left overnight at 4°C and centrifuged at 5,000 g (4°C) for 15 min. Plasma total cholesterol, triglyceride, high density lipoprotein-cholesterol, LDL-cholesterol (LDL-c) were measured using a colorimetric enzymatic kit including triglyceride, cholesterol reagents or HDL cholesterol precipitating reagent kits (ALPCO, Salem, NH, USA) according to the manufacturer's protocol.

Human THP-1 cells (ATCC, Manassas, VA, USA) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) medium and maintained at 37°C in humidified atmosphere containing 5% CO₂ in air and differentiation into adherent macrophages was induced by incubating at 37°C with 100 nM phorbol myristate acetate for 48 h. The medium was replaced with new medium containing 0, 5, 10 and 20 µM of luteolin for 24 h. Next, the cells were treated with 100 µg/ml ox-LDL (Alfa Aesar, Tewksbury, MA, USA) for 5 h at 37°C.

Total RNA was isolated from the mice proximal aorta or cultured cells with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. cDNA was synthesized by RT using reverse transcriptase (Takara Biotechnology Co., Ltd., Dalian, China). For reverse transcription, 2.5 µl of 20 µM primer stock was combined with RNA sample and heated with 70°C for 3 min. A total of 4 µl 5X First-Strand Buffer, 2 µl dNTP mix, 2 µl 100 mM DTT and 0.5 µl SMART MMLV reverse transcriptase were mixed and incubated at 42°C for 60 min. The reaction was terminated by heating at 70°C for 15 min. qPCR was performed on an iCycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using a SYBR PCR kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. The thermocycling conditions used were as follows: qPCR was performed at 42°C for 5 min, 95°C 10 sec for one cycle, followed by 40 cycles at 95°C for 5 sec, 60°C for 20 sec; and melting curve analysis was performed at 65°C for 15 sec, and increased to 95°C by 0.1°C/sec. Results were quantified using the 2^−∆∆Cq method (23). β-actin served as the housekeeping gene for the comparisons of the gene expression data. The primer sequences for qPCR are listed in Table I.

Following treatment, cells were lysed in radioimmunoprecipitation assay buffer containing 10 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology, Haimen, China). Total cellular protein (30 µg) determined by the BCA method was separated by 10% SDS-PAGE, transferred onto nitrocellulose membranes and blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 2 h at room temperature. Subsequently, the membrane was incubated with primary antibodies directed against SIRT1 (1:1,000; cat. no. 2028; Cell Signaling Technology, Inc., Danvers, MA, USA), AMPK (1:1,000; cat. no. 5832; Cell Signaling Technology, Inc.) and Thr172 phosphorylated-AMPK (pAMPK; 1:1,000; cat. no. 2535; Cell Signaling Technology, Inc.). Following incubation with the rabbit horseradish peroxidase-conjugated secondary antibodies (1:5,000, cat. no. 7074; Cell Signaling Technology, Inc.). Following incubation with the secondary antibodies, the immunoreactive bands were detected with an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.). Immunoblots were quantified using ImageQuant TL 7.0 software (GE Healthcare, Chicago, IL, USA) and expressed at a ratio of pAMPK to AMPK or SIRT1 to β-actin (1:1,000, cat. no. 4970; Cell Signaling Technology, Inc.).

Data are presented as the mean ± standard error of the mean. One-way analysis of variance with Duncan's post hoc tests were used for the mouse assays and in vitro assays P<0.05 was considered to indicate a statistically significant difference. SPSS 16.0 (SPSS, Inc., Chicago, IL, USA) was used as statistical software.

Plasma total cholesterol (P<0.001; Fig. 2A) and triglyceride (P<0.01; Fig. 2B) levels were significantly higher in mice fed with a western diet compared to the control group; however, total cholesterol (P<0.05; Fig. 2A) and triglyceride ~30% following luteolin treatment. Consistently, plasma LDL-c levels exhibited a 32.8% reduction in the luteolin treatment group compared with the western diet group (P<0.05; Fig. 2C). However, dietary luteolin had no significant effect on high-density lipoprotein cholesterol levels in mice fed with a western diet (Fig. 2D). These data indicate that luteolin ameliorates lipid accumulation in the plasma and prevents dyslipidemia in LDLR^−/− mice fed with a western diet.

The activation of monocytes and macrophages is an important initial step in atherosclerosis, sustaining inflammation within atheromasia and possibly influencing plaque stability (7–9). The macrophage recruitment and inflammation in the aortic root was therefore examined in the present study. RT-qPCR revealed that the macrophage marker cluster of differentiation 68 (CD68; P<0.01; Fig. 2E) and macrophage chemoattractant protein 2 (CCL2; P<0.05; Fig. 2F) were significantly reduced in the aortic root of mice fed a western diet supplemented with luteolin compared with mice fed with a western diet alone. Additionally, the levels of inflammatory cytokine interleukin (IL)-6 and TNF-α were significantly decreased in the aortic root following luteolin treatment (P<0.01; Fig. 2G and H). These results demonstrate that luteolin ameliorates atherosclerosis by decreasing macrophage infiltration into the plaque and inflammation in the aorta of LDLR^−/− mice.

In order to verify the effect of luteolin on macrophage inflammation in atherosclerosis, differentiated THP-1-derived macrophages were treated with increasing concentrations of luteolin. Incubation of THP-1 cells with 100 µg/ml ox-LDL significantly increased the uptake of lipids, as demonstrated by the higher total cholesterol level (P<0.05; Fig. 3A). Treatment with 10 or 20 µM of luteolin for 24 h significantly reduced the cholesterol content in THP-1 cells in a dose-dependent manner (P<0.05; Fig. 3A). Consistent with these results, luteolin also inhibited CCL2 (P<0.05; Fig. 3B), IL-6 (P<0.01; Fig. 3C) and TNF-α (P<0.01; Fig. 3D) expression in ox-LDL induced THP-1 cells in a dose-dependent manner. These results provide further evidence that luteolin decreases foam cell formation and inflammatory factors in macrophages, alleviating atherosclerosis.

AMPK-SIRT1 signaling has been reported to be associated with the attenuation of atherosclerosis and vascular dysfunction (21–25); luteolin may be able to activate this signaling in macrophages (26). In order to explore the effect of luteolin on lipid deposition and inflammation in macrophages, AMPK-SIRT1 signaling in luteolin-treated THP-1 cells was assessed. Western blot analysis revealed that 20 µM luteolin, an effective concentration for the inhibition of inflammation in THP-1 cells, significantly increased the levels of phosphorylated AMPK compared with cells without luteolin (P<0.05; Fig. 4A and B). Furthermore, luteolin significantly ameliorated the ox-LDL-induced decrease in SIRT1 expression in THP-1-derived macrophages (P<0.01; Fig. 4C). In aggregates, these results demonstrated that luteolin inhibits macrophage inflammation via a mechanism that is associated with activation of AMPK-SIRT1 signaling.