Cryopreserved HASMC were purchased from ScienCell Research Laboratory (San Diego, CA, USA). Cells were cultured as monolayers in smooth muscle cell medium (ScienCell) containing essential and nonessential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals, and 2 % (v/v) fetal bovine serum at 37 °C in a humidified 5 % CO2 atmosphere. Cells from passages 2 to 6 were used in this study.
Experimental animals and diet
Male ApoE−/− mice in a C57BL/6 N background (6 weeks of age) were from the Jackson Laboratory (Bar Harbor, ME, USA). They were housed under diurnal lighting conditions and allowed food and tap water ad libitum. All experimental protocols involving use of animals were conducted in accordance with the National Institutes of Health guidelines and approved by the Committee on Animal Care of KIOM.
The mice fed on a High Fat diet (45 % of total calories from fat; 0.15 % cholesterol; Research Diet, New Brunswick, NJ, USA) were divided into five groups: control (chow diet, n = 8), HFD (High Fat Diet, n = 8), Illicium v. 100 mg/kg treatment (HFD with Illicium v. 100 mg/kg, n = 8), Illicium v. 200 mg/kg (HFD with Illicium v. 200 mg/kg, n = 8), and atorvastatin 10 mg/kg (HFD with atorvastatin 10 mg/kg, n = 8). Daily treatments were given orally over a 12-week period, during which experimental animals were fed a HFD.
Preparation of Illicium verum extract
Illicium v. material was purchased from Omniherb Co. (Yeongcheon, South Korea) and was authenticated based on its microscopic and macroscopic characteristics by the Classification and Identification Committee of the KIOM. A voucher specimen has been deposited at the herbarium of the Basic Herbal Medicine Research Group at KIOM. Dried fruit (300 g) was extracted twice with 70 % (v/v) ethanol (with a 2-h reflux). The extract was concentrated under reduced pressure at 40 °C with a rotary evaporator. The decoction was filtered, lyophilized, and stored at 4 °C until use (Sung et al. ). The yield of the dried extract from the starting crude materials was approximately 15.73 % (w/w). The lyophilized powder was dissolved in 0.05–0.1 % dimethyl sulfoxide and then filtered through a 0.22 μm syringe filter to create a stock solution.
A standard assay based on the tetrazole (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to assess cell viability. Briefly, HASMCs were seeded in 96-well microtiter plates at a density of 1 × 105 cells/well. They were treated with various concentrations of Illicium v. (10, 50, or 100 μg/mL) for 24 h. Subsequently, 100 μL of 5 mg MTT/mL in PBS was added to each well, and the plates were incubated at 37 °C for 4 h before adding 200 μL DMSO to dissolve formazan crystals. Absorbance was measured at 540 nm by spectrophotometry.
NF-κB activity assay
Activity of NF-κB was measured by luciferase reporter assays in HASMC. Cells in 12-well plates were cotransfected with a firefly luciferase gene tagged with renilla luciferase and pGL4.32-NF-κB, using the FuGENE HD reagent (Invitrogen; Carlsbad, CA, USA). Medium was replaced with fresh medium after 6 h. At 24 h post transfection, cells were stimulated with TNF-α (10 ng/mL, R&D Systems Inc.; St. Louis, Mo, USA) in the presence of 10, 50, or 100 μg Illicium v../mL. Luciferase activity was assayed 24 h later using a dual-luciferase reporter assay system (Promega; Madison, Wl, USA).
Measurement of serum markers
Blood was collected from the aorta under light anesthesia and stored on ice for 30 min before centrifugation at 13,000 rpm at 4 °C for 10 min, and the serum was separated and kept at −80 °C until it was thawed for the assay. Serum levels of total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine were measured with an automatic analyzer.
Blood pressure measurement
Blood pressure was monitored using a noninvasive tail-cuff CODATM system (Kent Scientific; Torrington, CT, USA) as previously described .
Mice were deeply anesthetized with sodium thiopental and subsequently the tissue was removed and then washed with cold PBS and followed fixation with 4 % (w/v) paraformaldehyde. The aorta of each mouse was then removed and further fixed in the same solution for 24 h at 4 °C. Fixed aortas were frozen and stored at −80 °C. Sections of 10-μm thickness were sliced from frozen tissue and then immunostained with antibodies against iNOS(Thermo Scientific; Rockford, IL USA). After additional incubation with secondary antibody, sections were reacted with 3-amino-9-ethylcarbazole chromogen (Vector Laboratories; Burlingame, CA, USA). Reactions with 3,3′ diaminobenzidine substrate (Vector Laboratories) were performed for color development, and the stained sections were then analyzed by light microscopy.
Western blot analysis
Proteins were isolated from aortic tissue according to standard techniques , separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Amersham Biosciences; Piscataway, NJ, USA). Blots were probed with primary antibodies directed against VCAM-1 or NF-κB (Santa Cruz Biotechnology; San Cruz, CA, USA), or against ICAM-1, E-selectin, TNF-α, IL-1β, COX, IκB-α, or Iκκ-α/β, iNOS (Cell Signaling Technology; Beverly, MA, USA). This was followed by incubation with secondary antibody conjugated to horseradish peroxidase (Cell Signaling). Bound secondary antibody was detected by chemiluminescence generated by peroxidase reaction, measured with an ImageQuant LAS 4000 apparatus (GE Healthcare Life Sciences; Buckinghamshire, UK). Membranes were reprobed with an anti-β-actin antibody (Sigma-Aldrich Chemical Co.; St. Louis, MO, USA) as internal sample control.
The data are expressed as mean values ± SEM. Statistical comparisons were performed using analysis of variance for repeated measures, followed by Sigmastat statistical program Version 11.2 (Systat Software, San Jose, CA, USA). Data were analyzed statistically using two-way ANOVA via Tukey’s post hoc comparison when comparing more than two groups. A value of P <0.05 was considered to be statistically significant.