Male C57BL/6 (B6) and ICR mice (weight, 20–30 g) were purchased from Orient Bio, Inc. (Seongnam, Korea). The mice were maintained under specific pathogen-free conditions at the Catholic Research Institute of Medical Science of the Catholic University of Korea (Seoul, Korea), and were fed standard mouse chow and water. The mice were maintained in Plexiglass cages at a constant temperature of 22±2°C and a relative humidity of 55±5%, with a 12 h dark-light cycle for at least 1 week prior to the experimental session. All experimental procedures were approved by the Animal Research Ethics Committee of the Catholic University of Korea.

EOCO used in the present study was obtained from Fosto Company (Seoul, Korea). EOCO was emulsified in dimethyl sulfoxide (DMSO; 1:1, v/v; Sigma-Aldrich, St. Louis, MO, USA) and was administered intraperitoneally (i.p.) at two different concentrations (5 or 10 mg/kg) in distilled water. EOCO was administered 1 h prior to injection with one of the inflammation-inducing chemical agents: Carrageenan (n=7 per group) or thioglycollate (n=6 per group). The control mice were treated with the same volume of distilled water containing DMSO.

The initial hind paw thickness of the ICR mice was determined using a pocket thickness gauge (0–5 mm) (17). Each group of mice received subplantar administration of 50 µl carrageenan (2% w/v; Sigma-Aldrich) in saline into the right hind paw 1 h following injection with EOCO or vehicle. Mice in the control group (carrageenan-negative group) were injected with an equivalent volume of saline solution at the same time-point as carrageenan was administered to the other groups. Paw thickness was measured prior to the carrageenan injection, and at 1, 2, 3, 4 and 5 h following carrageenan injection. The degree of swelling induced at each time-point was calculated as the difference between the initial hind paw thickness and the thickness at the respective hour following carrageenan injection.

At 5 h, the mice were sacrificed by cervical dislocation and the skin tissues were removed from the carrageenan-injected right hind paws of each experimental group. Subsequent to rinsing in ice-cold phosphate-buffered saline (PBS), the tissue was homogenized, and the proteins were extracted from 150 mg tissue/ml PBS containing 0.4 M NaCl, 0.05% Tween 20, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM EDTA and complete protease-inhibitor cocktail (one tablet/50 ml; Roche Diagnostics, Indianapolis, IN, USA) at 4°C (18). Following centrifugation twice at 12,000 × g for 15 min at 4°C, the supernatant was stored at −70°C for the determination of cytokine levels.

Mice were injected intraperitoneally with 5 or 10 mg/kg EOCO, 1 h prior to thioglycollate injection. C57BL/6 mice were administered an i.p. injection of 2 ml sterile 3% thioglycollate (BD Biosciences, Franklin Lakes, NJ, USA), or PBS as a control (19). After 4 h, the mice were sacrificed by cervical dislocation, and the peritoneal cavity was flushed with 1.0 ml Hanks' buffered salt solution without Mg²⁺ and Ca²⁺ (Welgene, Seoul, South Korea). Following centrifugation at 1,000 × g for 5 min at room temperature, the supernatant was stored at −20°C for cytokine measurement by enzyme-linked immunosorbent assay (ELISA), and the pelleted peritoneal cells were collected in order to count the cell numbers. To collect more peritoneal cells, the peritoneal cavity was washed again with 10 ml Hanks' buffered salt solution. The collected peritoneal cells from each sample were resuspended in 5 ml Hanks' buffered salt solution, and the number of cells was determined manually with a hemocytometer using trypan blue (Gibco; Thermo Fisher Scientific, Inc.) (20).

The RAW 264.7 murine macrophage cell line was obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% heat inactivated fetal bovine serum (Wisent, Inc., St. Bruno, QC, Canada) and were maintained in a humidified 37°C incubator containing 5% CO₂.

The quantity of nitrite in the culture medium was measured as an indicator of NO production. For NO quantification, RAW 264.7 cells were seeded at a density of 5×10⁵ cells/well in 6-well plates and were pretreated with EOCO (1, 10, 50 or 100 µg/ml) for 1 h, followed by a 24 h incubation with LPS (1 µg/ml; Sigma-Aldrich), as described previously (21). Following 24 h of stimulation, 100 µl of each supernatant was collected, mixed with the same volume of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid), and incubated for 5 min at room temperature. The quantity of nitrite was determined by measuring the absorbance at a wavelength of 540 nm using an ELISA plate reader (Synergy™ MX, BioTek Instruments, Inc., Winooski, VT, USA). Nitrite concentration was calculated using standard solutions of sodium nitrite (NaNO₂).

RAW 264.7 cells (2×10⁴ cells/well) were seeded onto 96-well flat-bottom plates and were pre-incubated for 15–18 h. The cells were left untreated or were treated with various concentrations of EOCO (1, 10, 50 or 100 µg/ml), and were incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h. A total of 10 µl MTT (2.5 mg/ml; Sigma-Aldrich) was added to each well and the cells were incubated for a further 4 h. The MTT was then removed and the cells were lysed by the addition of 100 µl DMSO to each well. The optical density was measured at a wavelength of 540 nm using a Synergy™ MX microplate reader.

The expression levels of IL-1β, TNF-α and IL-6 were determined in paw homogenates, peritoneal fluid and the supernatant of lysed LPS-stimulated RAW 264.7 cells using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's protocols.

The stimulated RAW 264.7 cells were washed twice with PBS and lysed in lysis buffer [(0.5 M NaCl, 20 mM Tris-HCl, 0.25% Triton X-100, 1 mM ethylene glycol tetraacetic acid, 1 mM EDTA, 10 mM β-glycerophosphate, 10 mM NaF, 1 mM benzamidine, 300 µM Na₃VO_4, 2 µM PMSF, 1 mM dithiothreitol and one protease inhibitor cocktail tablet] on ice for 1 h, followed by centrifugation at 12,000 × g for 20 min at 4°C. The protein concentration of the lysates was determined using the Bradford dye-binding assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 50 µg protein from the supernatants was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Chalfont, UK) for 130 min at 100 V. Subsequently, the membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBS-T; 20 mM Tris, 500 mM NaCl, KCl, pH 7.5, and 0.05% Tween 20) for 2 h at room temperature. The membranes were then agitated in blocking buffer containing rabbit anti-iNOS immunoglobulin (Ig)G (1:1,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-8310), rabbit polyclonal anti-COX-2 (1:1,000; Cayman Chemical Company, Ann Arbor, MI, USA; cat. no. 160106) or mouse monoclonal anti-β-actin (1:10,000; ABM, Inc., Richmond, BC, Canada) antibodies overnight in a cold room. Membranes were washed with TBS-T at 10 min intervals for 30 min and were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-2004) or HRP-conjugated goat anti-mouse IgG (1:10,000; Santa Cruz Biotechnology, Inc., cat. no. sc-2005) for 2 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence detection system (GE Healthcare Life Sciences) on a LAS-4000 (Fujifilm, Tokyo, Japan). Densitometric values for each band were determined using ImageJ software, version 1.48 (National Institutes of Health, Bethesda, MD, USA), and were statistically analyzed.

The data are expressed as the mean ± standard error of the mean. Comparisons between the groups were performed with Mann-Whitney U-tests. All data were analyzed using SAS 9.1 software (SAS Institute, Cary, NC, USA) and P<0.05 was considered to indicate a statistically significant difference.

To determine the anti-inflammatory effects of EOCO, a carrageenan-induced paw edema mouse model was used. Mice were treated with EOCO (i.p., 5 or 10 mg/kg) or vehicle (control) 1 h prior to carrageenan injection, and carrageenan-induced paw thickness was measured. As presented in Fig. 1, paw thickness increased sharply 1 h after the subplantar injection of carrageenan, and consecutively increased until 5 h after the injection. Administration of 5 mg/kg EOCO (i.p.) significantly reduced paw edema 2 and 5 h following carrageenan injection, as compared with the control group (P<0.05). In addition, treatment with 10 mg/kg EOCO significantly inhibited carrageenan-induced paw edema 4 and 5 h following injection (P<0.05 and P<0.01, respectively).

To investigate the mechanism underlying the inhibitory effects of EOCO on the development of carrageenan-induced paw edema, the levels of proinflammatory cytokines in the paw tissue were determined. In the paw homogenates, the levels of IL-1β, IL-6 and TNF-α were significantly increased by carrageenan injection, as compared with saline (P<0.01; Fig. 2). The administration of 10 mg/kg EOCO markedly inhibited the carrageenan-induced increased levels of IL-1β and IL-6, as compared with the control group (P<0.05 and P<0.01, respectively). Conversely, 5 mg/kg EOCO significantly reduced IL-6 levels only. However, EOCO treatment did not affect the levels of TNF-α (Fig. 2C).

To investigate the anti-inflammatory effects of EOCO on a mouse model of peritonitis, mice were pretreated with EOCO (i.p.) 1 h prior to injection with 2 ml 3% thioglycollate (i.p.) to induce peritonitis. After 4 h, mice were sacrificed and the peritoneal fluid was collected. Total cells recruited into the peritoneal cavity following thioglycollate injection were markedly increased, as compared with in PBS-injected mice (Fig. 3A, P<0.01). Treatment with 5 or 10 mg/kg EOCO significantly reduced the number of total cells recruited into the peritoneal cavity, as compared with the control group (P<0.01 and P<0.05, respectively). In addition, the levels of IL-1β, IL-6 and TNF-α in the peritoneal lavage fluid were significantly reduced by EOCO treatment, as compared with the control group (Fig. 3B–D; P<0.05).

To investigate whether the anti-inflammatory effects of EOCO in thioglycollate-induced peritonitis were due to the inhibition of macrophage function, LPS-stimulated RAW 264.7 murine macrophage cells were treated with EOCO. RAW 264.7 cells were cultured with LPS (1 µg/ml) in the presence or absence of EOCO (1, 10, 50 and 100 µg/ml). EOCO decreased the production of NO, IL-6 and TNF-α by LPS-stimulated RAW 264.7 cells in a dose-dependent manner, and the inhibition was statistically significant at a dose of 100 µg/ml (P<0.001, P<0.01 and P<0.05, respectively; Fig. 4A–C). The cytotoxicity of EOCO on RAW 264.7 cells was determined using the MTT assay. Cells cultured in the presence of EOCO (1–100 µg/ml) for 24 h demonstrated no change in viability, as compared with those incubated in culture media only. However, cell viability was significantly decreased by ~78.8% following treatment with 200 µg/ml EOCO (Fig. 4D).

In order to investigate whether the inhibition of NO production was due to decreased iNOS and COX-2 protein expression, the effects of EOCO on iNOS and COX-2 protein expression levels were analyzed by western blotting. As presented in Fig. 5, the protein expression levels of iNOS and COX-2 in RAW 264.7 cells were significantly increased by LPS. The LPS-stimulated expression levels of iNOS and COX-2 proteins were significantly inhibited by treatment with 50 and 100 µg/ml of EOCO (Fig. 5).