MDMs were isolated from peripheral blood monocytes by adherence to plastic as described previously (30). Blood was layered onto Lymphoprep (Axis-Shield, Dundee, Scotland) and centrifuged for 30 min at 700 g. The white-blood-cell layer was harvested, washed with PBS and suspended in RPMI-1640. Cells were counted and then plated at 1×10⁶ cells per 140 mm dish in RPMI-1640 with 5% heat-inactivated human serum. After 2 h, the plates were washed three times in RPMI-1640 and then incubated at 37°C overnight. The cells were left to differentiate into MDMs for 7 days, then washed with PBS, treated with 5 mM PBS/EDTA at 37°C for 20 min, harvested gently with a cell scraper, counted and replated on 96- or 6-well trays at 1×10⁴ and 1×10⁶ cells per well, respectively, as described previously (31).

One hundred and twenty 8-week-old male Sprague-Dawley rats were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The animals were housed under standard conditions of a 12/12 h light/dark cycle at room temperature with a HFD (MD12033; Mediscience, Ltd., Jiangsu, China), and free access to water. All animal experimental procedures were conducted under the guidelines of the National Health and Medical Research Council for the Care and Use of Animals for Experimental Purposes in China. All efforts were made to minimize suffering.

MDMs were plated in triplicate into 12-well cell culture plates (Takara Biotechnology (Dalian), Co., Ltd., Dalian, China). The experimental regime consisted of cells undergoing a preconditioning phase of: i) 100 μl vehicle for 4 h; ii) 0.1 nmol berberine and 100 nmol atorvastatin for 4 h; iii) 1 nmol berberine and 100 nmol atorvastatin for 4 h; iv) 10 nmol berberine and 100 nmol atorvastatin for 4 h; v) 100 nmol berberine and 100 nmol atorvastatin for 4 h; vi) transfection with non-specific siRNA followed by the addition of 100 μl vehicle for 4 h; vii) transfection with non-specific siRNA followed by the addition of 100 nmol berberine and 100 nmol atorvastatin for 4 h; viii) transfection with specific siRNA targeting the ET-1 receptor followed by the addition of 100 nmol berberine and 100 nmol atorvastatin for 4 h. One hundred and twenty male Sprague-Dawley rats were randomly assigned to 6 groups and fed a HFD for 4 months prior to initiation of mimic atherosclerosis. Subsequently, the rats were exposed to treatment as follows: i) vehicle for 1 month (i.v.); ii) 0.1 μmol/kg berberine and 100 μmol/kg atorvastatin for 1 month (i.v.); iii) 1 μmol/kg berberine and 100 μmol/kg atorvastatin for 1 month (i.v.); iv) 10 μmol/kg berberine and 100 μmol/kg atorvastatin for 1 month (i.v.); v) 100 μmol/kg berberine and 100 μmol/kg atorvastatin for 1 month (i.v.); vi) 100 μg/kg/min BQ-788 (i.v.) followed by 100 μmol/kg berberine and 100 μmol/kg atorvastatin for 1 month (i.v.).

At the end of the treatment, body weight (BW) was measured and the animals were anesthetized with 10% chloral hydrate. Blood was collected by cardiac puncture. Organs such as heart, liver, kidneys and spleen were harvested and weighed.

Scrambled siRNA and siRNA targeting the ET-1 receptor were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Cells were transfected with scrambled or ET-1 receptor siRNA according to the manufacturer’s instructions. Briefly, the ET-1 receptor and scrambled siRNAs (30 pmol) were diluted in 500 μl DMEM and mixed with 5 μl Lipofectamine RNAi MAX (Invitrogen Life Technologies, Carlsbad, CA, USA). After 15 min incubation at room temperature, the complexes were added to the cells to a final volume of 3 ml medium. The cells were then harvested at the indicated times for further analysis. The efficiency of the ET-1 receptor siRNA was confirmed by western blot analysis of Flag expression.

Blood samples were collected and the levels of TC, TG, LDL-C, and HDL-C were detected with an automatic biochemistry analyzer (Hitachi, Tokyo, Japan). The samples were analyzed in duplicate.

The CRP levels were determined with an ultrasensitive CRP test with a coefficient of variance below 5% (Sigma, St. Louis, MO, USA). A Biochemical Analysis kit (Nanjing Jiancheng Biotechnology Co., Ltd., Nanjing, China) was used to measure MDA content, GPx, and SOD activity according to the manufacturer’s instructions.

The levels of ET-1 protein in serum were analyzed using a commercially available ELISA (Yanjin Biotechnology Co., Shanghai, China) according to the manufacturer’s instructions. The absorbance was read at 450 nm using a 680XR microplate reader (Bio-Rad, Hercules, CA, USA). All the samples were analyzed in duplicate. The standard curve for ET-1 estimation was conducted by linear regression analysis.

RNA was extracted from MDMs or monocytes using TRIzol RNA-extraction reagent (Gibco-BRL, Rockville, MD, USA) according to the manufacturer’s instructions. Total RNA (5 μg) for each sample was reverse transcribed into first-strand cDNA for qRT-PCR analysis. qRT-PCR was performed in a final volume of 10 μl, which contained 5 μl of SsoFast^TM EvaGreen supermix (Bio-Rad), 1 μl of cDNA (1:50 dilution), and 2 μl each of the forward and reverse primers (1 mM). The steps in qRT-PCR were performed as follows: 94°C for 2 min for initial denaturation; 94°C for 20 sec, 58°C for 15 sec, and 72°C for 15 sec; 2 sec for plate reading for 40 cycles; and a melt curve from 65 to 95°C. β-actin was used as a quantitative and qualitative control to normalize the gene expression. Data were analyzed using the formula: R = 2^{− [ΔCT sample−ΔCT control]}. All of the primers used in this experiment are shown in Table I.

Cells were homogenized and lysed with RIPA lysis buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% TritonX-100, 1 mM EDTA, 10 mM β-glycerophosphate, 2 mM sodium vanadate and protease inhibitor). Protein concentration was assayed using a micro-BCA protein kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Forty micrograms of protein per lane were separated by 12% SDS-PAGE and electroblotted onto nitrocellulose (Amersham Pharmacia Biotech, Freiburg, Germany). Non-specific binding was blocked by incubating with 5% non-fat milk in TBST buffer at room temperature for 1 h. Immunodetection of LOX-1 and β-actin was conducted using mouse monoclonal anti-LOX-1 antibody (1:1,000; Santa Cruz Biotechnology, Inc.), and anti-β-actin (Sigma), respectively. Goat anti-mouse IgG (1:5,000; Sigma) followed by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, NJ, USA) were used for the detection of β-actin.

Results are expressed as means ± SD. Statistical significance was analyzed with one-way factorial ANOVA or the Student’s two-tailed t-test. P<0.05 was considered statistically significant. Analyses were conducted using SPSS software (SPSS, Inc., Chicago, IL, USA).

To investigate the effect of berberine combined with atorvastatin on the expression of LOX-1, the two genes were analyzed by qRT-PCR and western blot analysis. The qRT-PCR results showed that the mRNA level of LOX-1 tended to decline as the amount of berberine increased by 10 to 1,000-fold (Fig. 1A). This result was confirmed by western blot analysis (Fig. 1B and C).

ET-1 has been shown to regulate the expression of LOX-1 through the ET-1 receptor in endothelial cells. To examine the involvement of the ET-1 receptor in the regulation of LOX-1 expression in MDMs, we transfected ET-1 receptor siRNA into MDMs. Following treatment with berberine and atorvastatin, the expression of LOX-1 mRNA was analyzed by qRT-PCR and western blot analysis. Transfection with siRNA significantly blocked the inhibitory effect of berberine combined with atorvastatin on LOX-1 mRNA expression (Fig. 2A), which was confirmed by western blot analysis (Fig. 2B and C).

To explore the effect of berberine combined with atorvastatin on the physiological parameters of the model rats, the BW, heart weight (HW), liver weight (LW), spleen weight (SW), and kidney weight (KW) of rats in different groups were calculated at the end of the treatment. BW, LW and KW were markedly increased at concentrations of berberine ≥1 μmol/kg, while HW and SW remained constant for all the groups. The BW gains were 7.6, 11.4 and 16.1%, the LW gains were 29.3, 43.9 and 48.8%, and the KW gains were 41.7, 62.5 and 66.7% in the 1, 10 and 100 μmol/kg berberine groups compared to the control (0 μmol/kg berberine group), respectively (Table II).

To investigate variations in serum lipid profiles in model rats treated with berberine and atorvastatin, serum TC, TG, LDL-C and HDL-C levels were monitored via an automatic biochemistry analyzer at the end of the treatment. Compared to the control group (0 μmol/kg), treatment with berberine in combination with atorvastatin notably decreased serum TC, TG and LDL-C levels in rats (Fig. 3A–C). However, no significant difference in the serum level of HDL-C was detected among rats in the different groups (Fig. 3D).

To validate whether treatment with berberine in combination with atorvastatin affected inflammation and oxidative stress in model rats, serum CRP, MDA, GPx and SOD were measured using commercial kits. The results showed that treatment with berberine in combination with atorvastatin distinctly reduced serum CRP and MDA levels and promoted serum GPx and SOD levels in the model rats (Fig. 4).

To explore variations in plasma ET-1 levels, ELISA was performed on rats in each group. Treatment provoked a marked decrease in the plasma ET-1 level compared to the control group (Fig. 5). Additionally, the expression of LOX-1 in monocytes was analyzed by qRT-PCR and western blot analysis. Compared to the control group, berberine in combination with atorvastatin significantly downregulated the expression of LOX-1 in monocytes (Fig. 6).

To examine whether the ET-1 receptor was involved in the regulation of LOX-1 expression by berberine and atorvastatin, model rats were preconditioned with an ET-1 receptor antagonist prior to oral uptake of berberine and atorvastatin. Compared to the control, treatment with berberine in combination with atorvastatin led to the downregulation of LOX-1 expression in monocytes. By contrast, ET-1 receptor antagonist preconditioning eliminated the inhibitory effect of berberine combined with atorvastatin and resulted in an increased expression of LOX-1 in monocytes (Fig. 7).