The chemicals used in the present study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against ICAM-1 (Cat. no. 4915), p65 (Cat. no. 8242), lamin A/C (Cat. no. 2032) and β-actin (Cat. no. 4967) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-VCAM-1 antibody (sc-8304) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Primary HASMCs were obtained from ScienCell Research Laboratories (San Diego, CA, USA). The cells were cultured as monolayers in smooth muscle cell (SMC) medium (ScienCell) containing essential and non-essential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals and 2% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. For subcultures, the cells were detached using 0.125% trypsin containing 0.01 M ethylenediaminetetraacetic acid (EDTA). The cells used in the present study were from the early passages (passages 2–6). THP-1 cells were from the American Type Culture Collection (ATCC; Manassas, VA, USA) and weres used for the cell adhesion assay with the HASMCs. These cells were cultured in RPMI-1640, and supplemented with 2 mM L-glutamine, 100 mg/ml streptomycin, 100 IU/ml penicillin and 10% FBS.

The root of C. wilfordii used in the present study was collected through KNRRC (Medicinal Plants Resources Bank NRF-2010-0005790) supported by the Korea Research Foundation (the resources of which were provided by the Ministry of Education, Science and Technology of Korea) in 2014. A voucher specimen (no. MPRBP00962) was deposited in the herbarium of Gachon University (Seongnam, Korea). The powder from the root of C. wilfordii (2,500 g) was extracted twice with 0–100% ethanol for 48 h at room temperature and the extract was concentrated under reduced pressure. The decoction was filtered, lyophilized and stored at 4°C until use.

CWE (100 g) was dissolved in distilled water and partitioned with n-hexane (Hx fraction), dichloromethane (CH₂Cl₂, MC fraction), ethyl acetate (EtOAc fraction), n-butanol (n-BuOH fraction) and water (H₂O fraction). The yield of dried extract from the starting crude materials was approximately 30.8% (wt/wt). The EtOAc fraction has a potent suppressive effect on the expression of the adhesion molecules, VCAM-1 and ICAM-1. Hence, the EtOAc fraction (2.55 g) was fixed on Celite, fractionated by vacuum liquid chromatography (VLC) on a silica gel, and eluted to 16 sub-fractions. Fractions 6, 8, 10 and 12 were subjected to silica gel column chromatography (CC) (150 g, 50×120 mm) and eluted with hexane/EtOAc/methanol (3:1:0.3) to yield 2,4-dihydroxyacetophenone (2,4-DHA), 2,5-dihydroxyacetophenone (2,5-DHA), p-hydroxyacetophenone (p-HA) and (Cyn A):

2,4-DHA: colorless solid; ¹H-NMR (500 MHz, CDCl₃): δ 7.72 (1H, d, J=7.04 Hz), 6.35 (1H, dd, J=2.5 and 7.04 Hz), 6.24 (1H, d, J=2.0 Hz), 2.51 (3H, s); ¹³C-NMR (125 MHz, CDCl₃): δ 114.5, 166.6, 103.6, 166.4, 109.2, 134.6, 204.3, 26.3. ii) 2,5-DHA: yellow powder; ¹H-NMR (500 MHz, CDCl₃): δ 7.21 (1H, d, J=2.24 Hz), 7.01 (1H, dd, J=2.5 and 7.04 Hz), 6.78 (1H, d, J=7.3 Hz), 2.58 (3H, s); ¹³C-NMR (125 MHz, CDCl₃): δ 120.8, 156.7, 119.7, 166.4, 126.0, 116.5, 206.0, 27.0.

The experimental animal facility and study protocols (GIACUC-R2013017) were approved by the Animal Care and Use Committee of Gachon University. All experimental procedures were undertaken in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA) and the National Animal Welfare Law of the Republic of Korea.

Four-week-old male C57BL/6 mice were obtained from Japan SLC Inc. (Shizuoka, Japan) and maintained in a controlled environment of 22±1°C and a humidity of 50±10% with a 12-h light-dark cycle for 1 week prior to the commencement of the experiments. Mice had access to sterile standard mouse chow and water ad libitum. At the start of the study, the diet was changed to an atherogenic (ATH) diet [1.25% (w/w) cholesterol, 0.5% (w/w) cholic acid and 16% (w/w) fats in the form of soybean oil, cocoa butter and coconut oil] or a normal control (NC) chow [0.3% (w/w) cholesterol, no cholic acid and 5% (w/w) fats]. Both diets were obtained from Research Diets Inc. (New Brunswick, NJ, USA).

The mice were divided randomly into 6 groups of 5 mice as follows: i) mice fed a normal control chow diet plus the vehicle (PBS; NC group); ii) mice fed an ATH diet plus the vehicle (PBS; ATH group); iii) mice fed an ATH diet plus 50 mg/kg body weight (bw)/day of CWE; iv) mice fed an ATH diet plus 100 mg/kg bw/day of CWE; v) mice fed an ATH diet plus 200 mg/kg bw/day of CWE; and vi) mice fed an ATH diet plus 10 mg/kg bw/day of simvastatin (Simv; Sigma-Aldrich) via oral gavage for 12 weeks. At the end of the treatments, each mouse was anesthetized and the thorax was opened. The aorta was dissected following perfusion with phosphate-buffered saline (PBS) and stored at −80°C until RNA isolation.

The HASMCs were seeded in 96-well flat-bottom plates (2×10⁴ cells/well) and then treated with CWE (2, 20 and 200 μg/ml) for 16 h. The cells were incubated with 100 μl of 5 mg/ml MTT [3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide] (Sigma-Aldrich) for a further 2–4 h. After the supernatant was removed, 100 μl of DMSO per well was added to the cells and mixed on a shaker for 15 min to dissolve the formazan crystals formed. The optical density (OD) colored solution was quantified at a 570 nm wavelength using an enzyme-linked immunoabsorbent assay (ELISA) reader (model 550 microplate reader, Bio-Rad Laboratories, Hercules, CA, USA).

The adhesion of THP-1 cells to the HASMCs was measured as previously described (18). Briefly, the HASMCs (which were grown in 96-well plates) were pre-treated with CWE (2, 20 and 200 μg/ml) for 2 h at 37°C. The cells were washed with medium and incubated with fresh growth medium containing TNF-α (10 ng/ml) for 8 h. The medium was removed from the wells and calcein AM-labeled THP-1 cells (2×10⁵ cells/ml) in 0.2 ml of the medium were added to each well. The test and control samples were used in triplicate in each experiment. Following incubation for 1 h in 5% CO₂ at 37°C, micro-wells were washed twice with 0.2 ml of warm medium. The number of adherent cells was detected using a fluorescence microscope (IX71; Olympus, Tokyo, Japan) equipped with a digital camera (DP71; Olympus) and processed using ImageJ software version 1.45s (National Institutes of Health). The increase in the adhesion of THP-1 cells upon stimulation of the HASMCs with TNF-α was calculated in relation to the basal adhesion of THP-1 cells to the unstimulated HASMCs (which was set to 1).

The cells were pretreated with CWE (2, 20 and 200 μg/ml) or dexamethasone (50 ng/ml) for 2 h. The cells were washed with medium and incubated with fresh growth medium containing TNF-α (10 ng/ml) for 30 min or 8 h. Following treatment, the cells were washed twice with PBS and lysed in ice-cold lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5% (v/v) NP-40, 0.1% (w/v) sodium dodecyl sulfate (SDS)] containing protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA) for 1 h. The lysates were then collected after centrifugation at 1500 × g for 10 min at 4°C. Cytosolic and nuclear extracts were prepared using a Nuclear Extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. The protein concentration was determined using a protein assay kit (Bio-Rad Laboratories) with bovine serum albumin (BSA) as the standard. Protein lysates (20 μg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred by electrophoretic means to an Immobilon^®-P Polyvinylidene difluoride membrane (Amersham, Arlington Heights, IL, USA) and probed with appropriate antibodies. The blots were developed using an enhanced chemoluminescence (ECL) kit (Amersham). In all the westernt blotting experiments, the blots were re-probed with anti-β-actin antibody as a control for protein loading.

Total RNA was extracted using a single-step guanidinium thiocyanate-phenol-chloroform method. The yield and purity of the RNA were confirmed by measuring the ratio of the absorbance values at 260 and 280 nm. PCR was undertaken using ICAM-1- and VCAM-1-specific primers to identify their respective specific cDNA. The following sequence-specific primers were synthesized: 5′-ATTTTCTGG GGCAGGAAGTT-3′ and 5′-ACGTCAGAACAACCGAAT CC-3′ for human VCAM-1; 5′-AGCACCTCCCCACCTAC TTT-3′ and 5′-AGCTTGCACGACCCTTCTAA-3′ for human ICAM-1. The following pair of oligonucleotides was used as the internal control: 5′-AACTTTGGCATTGTGGAAGG-3′ and 5′-ACACATGGGGGTAGGAACA-3′ for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The absence of contaminants was routinely checked by an RT-PCR assay of negative control samples without the addition of a primer. Following amplification, the samples were stored at −20°C.

An Alliance 2695 system (Waters Corp., Milford, MA, USA) coupled with a Waters 2998 photodiode array detector was used for the quantitative chromatographic analysis of CWE. The analytical column was a Sunfire™ 4.6 × 150 mm C18 column (particle size, 5 μm; Waters Corp.). The mobile phase consisted of (A) acetic acid (0.5% v/v) and (B) acetonitrile using a gradient elution of A/B = 90/10 (0 min) → A/B = 65/35 (10 min) → A/B = 0/100 (30 min). The flow rate was 1.0 ml/min and the injection volume was 10 μl; ultraviolet (UV) detection was conducted at 254 nm. CWE was dissolved in ethanol at 10 mg/ml, and p-HA (purity 99%), 2,4-DHA (purity 99%) and 2,5-DHA (purity 97%) were used as standard solutions.

Each result is reported as the mean ± SEM. One-way analysis of variance was used to determine significance among groups, after which the modified Student’s t-test with the Bonferroni correction was used for the comparison between individual groups. A value of P<0.05 was considered to indicate a statistically significant difference.

We investigated the effects of the ethanol concentration for the root extract of C. wilfordii on the inhibition of the expression of adhesion molecules in the TNF-α-stimulated HASMCs. Ten different concentrations of ethanol (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100%, v/v) were used by adjusting the composition of ethanol and water in the extraction solvent. The cells were pre-treated with 100 μg/ml of each ethanol extract and then incubated with fresh growth medium containing TNF-α (10 ng/ml) for 8 h. We found that the ethanol extracts obtained with various concentrations had suppressive effects on TNF-α-induced VCAM-1 expression in the HASMCs (Fig. 1). Among these, the most marked inhibitory effect on VCAM-1 expression was observed by treatment with an ethanol concentration of 90% (Fig. 1). The extracts obtained at low or high ethanol concentrations (10, 20 and 100%) had lower efficacy, but they were comparable with the cells treated with dexamethasone (50 ng/ml).

We determined the effects of CWE on the TNF-α-induced expression of adhesion molecules in HASMCs. Western blot analysis produced the following results i) TNF-α significantly induced the expression of VCAM-1 and ICAM-1; and ii) CWE downregulated the TNF-α-induced expression of the adhesion molecules in a dose-dependent manner (Fig. 2A and B).

Moreover, MTT assay revealed that CWE did not affect cell viability and was not cytotoxic to the cells at the concentrations used (Fig. 2C).

We determined the effects of CWE on the adherence of THP-1 monocytes to TNF-α-stimulated HASMCs. The HASMCs were treated without or with various concentrations (2, 20 and 200 μg/ml) of CWE for 2 h prior to stimulation with TNF-α (10 ng/ml). Stimulation with TNF-α elicited a significant increase in the adhesion of THP-1 monocytes to the HASMCs (P<0.01). Treatment with CWE significantly inhibited the adhesion of the THP-1 monocytes to the HASMCs in a dose-dependent manner (Fig. 3).

To verify the in vitro effects of CWE, an in vivo experiment was undertaken using a mouse model of ATH diet-induced hypercholesterolemia. RT-PCR revealed the expected significant increase in the mRNA expression of VCAM-1 and ICAM-1 in the aortas of the hypercholesterolemic mice. The administration of CWE for 12 weeks dose-dependently reduced the expression of VCAM-1 and ICAM-1 in the aortaso the hypercholesterolemic mice. The suppressive effects of CWE (100 and 200 mg/kg) on the expression of CAMs were comparable to those observed by treatment with Simv (Fig. 4).

NF-κB is a crucial transcription factor for the induction of the expression of adhesion molecules by TNF-α (19,20). Therefore, we investigated whether the inhibitory effects of CWE on the TNF-α-induced expression of ICAM-1 and VCAM-1 are mediated by the activation of NF-κB. The cells were treated with CWE (2, 20 and 200 μg/ml) for 2 h prior to stimulation with TNF-α for 30 min. CWE decreased the translocation of NF-κB p65 to the nuclear fraction in a dose-dependent manner (Fig. 5). These data suggested that CWE inhibited the TNF-α-induced nuclear translocation of NF-κB.

We performed solvent fractionation of CWE and evaluated the effects of the fractions on the expression of adhesion molecules to select the most promising fraction (Fig. 6). RT-PCR revealed that the fraction with ethyl acetate (EtOAc) inhibited the mRNA expression of VCAM-1 and ICAM-1 by approximately 80 and 40% in the TNF-α-stimulated HASMCs, respectively (Fig. 7A and B). The EtOAc fraction was found to be more active with lower cytotoxicity (Fig. 7C) than the other fractions.

As described above, the EtOAc fraction had the maximum inhibitory effect on the expression of VCAM-1 and ICAM-1 and did not elicit cytotoxicity. Hence, we investigated the dose-response effects of this fraction on the expression of VCAM-1 and ICAM-1 in the TNF-α-stimulated HASMCs. The EtOAc fraction markedly inhibited the TNF-α-induced mRNA expression of VCAM-1 and ICAM-1 in a dose-dependent manner (Fig. 8).

Next, we investigated whether 4 major acetophenones from the EtOAc fraction of CWE (p-HA, 2,4-DHA, Cyn A and 2,5-DHA) inhibit the TNF-α-induced expression of VCAM-1 and ICAM-1 in the HASMCs. Among these components, p-HA and Cyn A significantly inhibited the mRNA expression of VCAM-1 and ICAM-1 at 10 and 50 μg/ml (Fig. 9). However, treatment with 2,4-DHA and 2,5-DHA had little or no effect on the expression of VCAM-1 and ICAM-1. These 4 components did not affect cell viability at the concentrations tested (data not shown).

We applied HPLC for the simultaneous quantification of p-HA, 2,4-DHA, Cyn A and 2,5-DHA in CWE. The levels of p-HA, 2,4-DHA, Cyn A and 2,5-DHA identified at the retention times of 11.22, 12.49, 12.80 and 13.23 min were 3.8, 4.0, 21.0 and 1.0 mg/g, respectively (Fig. 10).