ARE was provided by the Dalian Institute of Chemical Physics, Chinese Academy of Sciences (Dalian, China), and dissolved and diluted in incubation medium to yield final concentrations of 30‐120 μg/ml. TNF-α was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Goat monoclonal anti-VCAM-1, goat monoclonal anti-ICAM-1, mouse monoclonal NF-κB p65, mouse monoclonal phosphorylated inhibitor of κB (p-iκB)-α, goat polyclonal Lamin B and anti-β-actin antibodies were purchased from Santa Cruz (CA, USA). Rat anti-mouse Mac3 and rat anti-mouse CD31 antibodies were purchased from BD Pharmingen (San Diego, CA, USA). The mouse soluble VCAM-1 immunoassay and mouse soluble ICAM-1 immunoassay kits were purchased from R&D Systems (MN, USA). The enhanced chemiluminescence (ECL) western blotting system was from Amersham Biosciences (Buckinghamshire, UK).
Endothelial cell culture and treatment
The murine endothelial cell line (SVEC4-10) was purchased from the American Type Culture Collection (MD, USA) and cultured in DMEM supplemented with 10 % fetal calf serum (FCS, HyClone, UT, USA), 30 μg/ml endothelial cell growth factor (Sigma-Aldrich, Saint Louis, MO, USA), 5 U/ml heparin, 100 U/ml penicillin, and 100 U/ml streptomycin at 37°C in an atmosphere of 5 % CO2 and 95 % air. The phenotype of SVEC cells was confirmed morphologically and by positive immunofluorescence staining of CD31. Cells were treated with 30, 60, or 120 μg/ml ARE in fresh serum-free medium and incubated for 2, 4, 6, or 8 h.
Culture of THP-1 cells
THP-1 cells were purchased from the American Type Culture Collection and grown in RPMI-1640 media (Sigma) containing 10 % FCS, 100 μg/ml streptomycin, 100 IU/ml penicillin, 250 ng/ml fungizone, 1 mM glutamine, 5 × 10–5 M 2-mercaptoethanol, and routinely subcultured three times per week at a ratio of 1:5.
Male apoE−/− mice (n = 24, age 8 weeks, 18–20 g) on a C57BL/6 J background were obtained from the Department of Laboratory Animal Science, Peking University Health Science Center, China. All mice were barrier housed, specific pathogen-free, and maintained in static microisolator cages. Mice were fed a high-fat, high-cholesterol diet containing 15 % fat and 0.25 % cholesterol. ApoE−/− mice were randomized into either a control group or an ARE group (n = 12 for each group). ARE mice were given oral doses of ARE (5 g/kg body weight per day). Blood and tissues were collected at 24 weeks of age for further analysis. The care and use of animals were performed in adherence with the National Institutes of Health Guidelines, and all experimental procedures involving animals were approved by the Ethics Committee of Shenyang Northern Hospital.
Adhesion assay for THP-1 cells
SVEC cells were plated in six-well plates and allowed to grow to confluence, as described above. The medium was removed and the cells were then incubated with 1.0 ml/well DMEM medium containing 10 % FCS with or without TNF-α (10 ng/ml) or TNF-α plus ARE at the concentrations and incubation times indicated above. Culture supernatant was removed from the treated cells at the indicated time points, and the cells were gently washed three times with DMEM medium. A volume of 1.0 ml of RPMI-1640 media containing 1 × 106 THP-1 cells was then added to the endothelial cell monolayers in each well. The binding phase of the assay was performed at 37°C in a 5 % CO2 atmosphere for 15 min for THP-1 cells. Thereafter the wells were washed three times with phosphate-buffered saline (PBS) (1.0 ml/well), and all wells were examined under a microscope to determine if any loss of endothelial cells had occurred during incubation or washing. The number of THP-1 cells washed in PBS was counted using a counting slide. Experiments were performed in triplicate.
Enzyme-linked immunosorbent assay (ELISA)
Aliquots of culture medium were collected from cells treated as described above. VCAM-1 and ICAM-1 levels in the culture medium were measured directly using an ELISA kit (R&D Systems), according to the manufacturer’s instructions. Standards containing known amounts of recombinant VCAM-1 and ICAM-1 were also analyzed. ELISA assay results were measured using a microplate reader. Absorbance was measured spectrophotometrically at 450 nm and plotted against a standard curve with VCAM-1 and ICAM-1 levels expressed in ng/ml. Experiments were performed in triplicate.
Western blot analysis
Confluent cells were cultured in medium with or without TNF-α (10 ng/ml) or TNF-α plus ARE at the concentrations and the incubation times indicated above to detect VCAM-1, ICAM-1, NF-κB, and p-iκB. Total proteins were extracted with 100 μl of lysis buffer (50 mmol/l Tris, 10 mmol/l MgCl2, 0.5 mol/l NaCl, 1 % Triton X-100) supplemented with a protease inhibitor mixture and 1 mmol/l phenylmethylsulfonyl fluoride. Cells were maintained on ice and lysates were harvested by scraping. The supernatants were collected after centrifugation at 14,000 g for 10 min. For nuclear protein analysis, cells were resuspended in hypotonic buffer on ice and detergent was added to 0.5 %. Nuclei were pelleted and resuspended in nuclear protein extract buffer (50 mmol/l HEPES [pH 7.8], 300 mmol/l NaCl, 50 mmol/l KCl, 0.1 mmol/l EDTA, 10 % [v/v] glycerol) on ice for 30 min and then spun, and the supernatant was saved. Protein levels in the supernatant were determined using the BCA method. Aliquots of 30 μg of each protein sample were resolved by 10 % sodium dodecyl sulfate polyacrylamide-gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After transfer, membranes were blocked with 5 % nonfat dry milk in Tris-buffered saline and then incubated overnight with primary antibodies at 4°C. After washing, blots were further incubated with 1:2,000 diluted secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-mouse, goat anti-rabbit, or rabbit anti-goat IgG for 2 h at room temperature. After additional washing, HRP activity was detected by ECL, according to the manufacturer’s instructions.
Quantitation of arterial atherosclerotic lesions
The proximal aortas of apoE−/− mice were immersed in ice-cold 4 % paraformaldehyde for 30 min. The samples were then transferred to 30 % sucrose-PBS solution at 4°C for 24 h, embedded in tissue freezing medium and snap-frozen in liquid nitrogen. Cryosections (5 μm thick) were cut from the proximal aortas beginning at the end of the aortic sinus, and stained with oil red O and hematoxylin and eosin. Arterial atherosclerotic lesions were also measured in aortas by en face oil red O staining. Quantitative analysis of the lesions was performed using Image Pro Plus software on at least 15 sections from each animal by an operator who was blinded to group assignment.
SVEC cells were plated on coverslips and incubated at 37°C and 5 % CO2 to allow the cells to adhere and spread. Serial 5-μm-thick cryosections were cut from the proximal aortas. Cells or cryosections were fixed with 4 % paraformaldehyde for 20 min. After washing three times with PBS, cells or cryosections were permeabilized with 1 % Triton X-100 in PBS for 30 min and blocked with goat serum for 20 min at room temperature. The cells were stained with primary antibodies against NF-κB. The cryosections were incubated with primary antibodies to VCAM-1, ICAM-1 or Mac-3. Alexa Fluor 488, 555, and 568 secondary antibodies (Invitrogen, OR, USA) were used at concentrations of 1:300. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Stained cells or tissues were observed using the Leica QWN (Wetzlar, Germany) analysis system.
Assessment of cell viability
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, Saint Louis, MO, USA) assay is based on the cleavage of MTT by mitochondrial dehydrogenases, reflecting the cell viability. SVEC cells were treated with 10 ng/ml TNF-α in the absence or presence of ARE (30, 60 or 120 μg/ml) for 8 h, followed by the addition of 0.5 mg/ml MTT medium and incubation for 4 h at 37°C. The medium was removed and dimethyl sulfoxide was added. The absorbance was examined at 570 nm.
The data are expressed as mean ± SD. All data were analyzed using SPSS 13.0 statistical software. Differences between two groups were compared using unpaired Student’s t-tests. Differences among three or more groups were compared using one-way analysis of variance (ANOVA). Statistical significance was defined as P < 0.05 (two-tailed).