RAW264.7 murine macrophage cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were incubated at 37°C in 5% CO₂ in Dulbecco's modified Eagle's media (DMEM; Gibco-BRL, Carlsbad, CA, USA), supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 5.5% fetal bovine serum (Gibco-BRL). The cells were also treated with 0.05% dimethyl sulfoxide as a vehicle control. The cells were seeded in 96-well plates at a density of 5×10⁴ cells/well and incubated in serum-free medium in the presence of different concentrations of TO. Following a 24 h incubation, the cellular viability was determined using an MTT assay. TO was obtained from the Plant Extract Bank at the Korea Research Institute of Bioscience and Biotechnology (PB1411.1; Daejeon, South Korea). All experiments were performed in triplicate. TO concentrations were used that have been shown to be nontoxic in other biological systems, based on MTT results (Fig. 1A).

The cells (2.5×10⁵ cells/ml) were seeded in 96-well plates in phenol red-free DMEM, and treated with different concentrations (10, 20, 30, and 40 µg/ml) of TO for 1 h, followed by an incubation in the presence of lipopolysaccharide (LPS; 1 µg/ml) for 24 h. Nitrite accumulation in the culture medium was measured using Griess reagent (Promega Corporation, Madison, WI, USA). The absorbance was measured at 535 nm, using a microplate reader (Bio-Rad, Hercules, CA, USA).

Total RNA was isolated from the cells using TRIzol^® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). A total of 2 µg of RNA was reverse transcribed, to synthesize single-stranded cDNA, using the commercially available kit (Qiagen, Valencia, CA, USA). The cDNA was then subjected to PCR. The primer sequences (Bioneer, Daejeon, Korea) used were as follows: tumor necrosis factor (TNF)-α, sense, 5′-GTG GAA CTG GCA GAA GAG GC-3′, antisense, 5′-AGA CAG AAG AGC GTG GTG GC-3′; COX-2, sense, 5′-CAA GTC TTT GGT CTG GTG CCT G-3′, antisense, 5′-GTC TGC TGG TTT GGA ATA GTT GC-3′; iNOS, sense, 5′-CAA GAG TTT GAC CAG AGG ACC-3′, antisense, 5′-TGG AAC CAC TCG TAC TTG GGA-3′; interleukin (IL)-6, sense, 5′-GAG GAT ACC ACT CCC AAC AGA CC-3′, antisense, 5′-AAG TGC ATC ATC GTT GTT CAT ACA-3′; MMP-9, sense, 5′-AAG CAC ATG CAG AAT GAG TAC CG-3′, antisense, 5′-GTG GGA CAG CTT CTG GTC GAT-3′; and β-actin, sense, 5′-CGC TCA TTG CCG ATA GTG AT-3′; and antisense 5′-TGT TTG AGA CCT TCA ACA CC-3′. The PCR products were fractionated and analyzed qualitatively by 1.5% agarose gel electrophoresis, stained with 5 µg/ml ethidium bromide for visualization.

The cells (2.5×10⁵ cells/ml) were seeded into 96-well plates in phenol red-free DMEM and treated with different concentrations (10, 20, 30 and 40 µg/ml) of TO for 1 h, followed by an incubation in the presence of LPS for 24 h. The cell supernatants were collected and were loaded for gelatin zymography. SDS−PAGE zymography was performed according to the methods of Heussen and Dowdle (15), to determine gelatinase activities. Briefly, zymogram gels, consisting of 10% polyacrylamide gel containing SDS and 1% gelatin, were used as the MMP substrate. The gels were washed in 2.5% Triton X-100 for 1 h to remove the SDS, followed by an incubation at 37°C for 16 h in developing buffer (1 M Tris-HCl, pH 7.5 with CaCl₂). The gels were subsequently stained with 25% methanol/8% acetic acid, containing Coomassie Brilliant Blue (Amresco, Solon, OH, USA). Gelatinase activity was visualized as white bands on a blue background, this represented the areas of proteolysis.

Female BALB/c specific pathogen-free mice (six weeks old) were purchased from Koatech Co. (Pyeongtaek, South Korea). Experimentation began after a two week quarantine and acclimatization period. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology. The mice were sensitized on days 0 and 14, by an intraperitoneal (i.p) injection of 20 µg OVA (Sigma-Aldrich, St. Louis, MO, USA) emulsified with 2 mg aluminum hydroxide in 200 µl phosphate-buffered saline (PBS) buffer (pH 7.4). On days 21, 22, and 23, the mice received airway challenges of OVA (1% (w/v)) for 1 h using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan). TO and montelukast, which was used as a positive control, were orally administered to the mice at a dose of 30 mg/kg body weight, one hour prior to OVA challenge. Airway responsiveness was indirectly assessed 24 h after the final challenge, using single-chamber, whole body plethysmography (Allmedicus Co. Ltd., Seoul, South Korea). The mice were sacrificed 48 h after the final challenge, by i.p injection of pentobarbital (50 mg/kg; Hanlim Pharm. Co. Ltd., Seoul, South Korea), and tracheostomies were performed. To obtain bronchoalveolar lavage fluid (BALF), ice-cold PBS (0.5 ml) was infused into the lung three times, and the fluid was withdrawn each time by tracheal cannulation (total volume 1.5 ml). Total inflammatory cell numbers were determined by counting the cells in ≤5 squares of a hemocytometer, following the exclusion of dead cells, with Trypan blue staining. Differential BALF cell counts were performed using Diff-Quik^® staining reagent (IMEB Inc., San Marcos, CA, USA), according to the manufacturer's instructions.

The levels of IL-4, IL-5, and IL-13 in the BALF were measured using ELISA kits (R&D SystemS, Minneapolis, MN, USA) according to the manufacturer's instructions. The levels of total IgE and OVA-specific IgE in the serum were also measured by ELISA. Microtiter plates were coated with anti-IgE antibodies (anti-mouse IgE; 10 g/ml; Serotec, Oxford, UK), in PBS-Tween^® 20, and incubated with BALF or a plasma sample. The plates were then washed four times with wash solution (PBS, containing 0.05% Tween 20 (Biosesang, GyeongGi-Do, Korea), and 200 µl of o-Phenylene-diamine dihydrochloride (Sigma-Aldrich) was added to each well. The plates were incubated for 10 min in the dark, and the absorbance was measured at 450 nm.

The murine lung tissue was homogenized (1/10 w/v) using a homogenizer with Tissue Lysis/Extraction reagent (Sigma-Aldrich), containing a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using a protein assay reagent (Bio-Rad), according to the manufacturer's instructions. Equal amounts of total cellular protein (30 µg) were separated by 12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). The membranes were incubated with blocking solution (5% skim milk; Becton Dickinson, Franklin Lakes, NJ, USA), followed by an overnight incubation at 4°C with the appropriate primary antibody. The following primary antibodies and dilutions were used: anti-β-actin (1:2,000 dilution; Cell Signaling Technology Inc., Danvers, MA, USA), anti-iNOS (1:1,000 dilution; Abcam, Cambridge, MA, USA) and anti-MMP-9 (1:1,000 dilution; Cell Signaling Technology Inc.). The blots were washed three times with Tris-buffered saline containing Tween^® 20 (TBST), followed by an incubation with a 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., Westgrove, PA, USA) for 30 min at room temperature. The blots were washed three additional times with TBST, and visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific, Waltham, MA, USA). Densitometic values for each protein were measured using a Chemi-Doc system (Bio-Rad).

After the BALF samples had been obtained, the lung tissues were fixed in 4% (v/v) paraformaldehyde. The tissues were embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin & eosin solution (Sigma-Aldrich) and Periodic acid-Schiff (IMEB Inc) in order to estimate inflammation and mucus production, respectively.

The data are expressed as the means ± standard error of the mean. Statistical significance was determined using analyses of variance, followed by multiple comparison tests with Dunnet's adjustment A P<0.05 was considered to indicate a statistically significant difference.

Treatment with TO did not induce toxicity in RAW264.7 murine macrophage cells at concentrations ≤40 µg/ml (Fig. 1A). NO production was significantly increased in the LPS-stimulated RAW264.7 cells, as compared with the control cells. Conversely, the TO-treated cells exhibited concentration-dependent reductions in NO production, as compared with the LPS-stimulated RAW264.7 cells (Fig. 1B).

The LPS-stimulated RAW264.7 cells exhibited increased relative mRNA expression levels of iNOS, COX2, IL-6, TNF-α and MMP-9. However, TO-treated cells exhibited a significant reduction in the relative mRNA expression levels of these proinflammatory mediators, as compared with the LPS-stimulated cells (Fig. 2A). Furthermore, MMP-9 activity was increased in the LPS-stimulated RAW264.7 cells, whereas reductions were observed in the TO-treated cells (Fig. 2B).

AHR was significantly increased in the OVA-sensitized/challenged mice, as demonstrated by an increased concentration of methylcholine, as compared with the normal controls. The AHR, induced by the OVA challenge, was reduced in the positive control montelukast-treated mice. The TO-treated mice also exhibited significant reductions in AHR, as compared with the OVA-sensitized/challenged mice (Fig. 3).

The OVA-sensitized/challenged mice exhibited a significant increase in the number of inflammatory cells present in the BALF, relative to the normal control mice (Fig. 4). However, the TO-treated mice exhibited significant reductions in the number of inflammatory cells, particularly eosinophils, in the BALF, as compared with the OVA-sensitized/challenged mice.

The OVA-sensitized/challenged mice showed marked elevations of Th2 cytokines, including IL-4, IL-5, and IL-13, in the BALF as compared with the normal controls. The elevations of Th2 cytokines induced by an OVA-challenge in the TO-treated mice were significantly reduced (Fig. 5). The production of eotaxin was also significantly increased in the OVA-sensitized/challenged mice; however, eotaxin production in the TO-treated mice was markedly reduced, as compared with the OVA-sensitized/challenged mice.

Total IgE was markedly elevated in the OVA-sensitized/challenged mice, as compared with the normal controls (Table 1). However, the TO-treated mice exhibited a significant reduction in the total serum IgE levels, as compared with the OVA-sensitized/challenged mice. These findings were consistent with the OVA-specific IgE serum results. The OVA-sensitized/challenged mice exhibited significantly increased OVA-specific IgE levels in the serum, whereas the TO-treated mice exhibited marked reductions, as compared with the OVA-sensitized/challenged mice.

Lung sections from the OVA-sensitized/challenged mice were examined for inflammatory cell infiltration into the peribronchial and perivascular lesions. The TO-treated mice exhibited reductions in airway inflammation, as compared with the OVA-sensitized/challenged mice (Fig. 6). Mucus production was also increased in the bronchial airways of the OVA-sensitized/challenged mice. Conversely, the TO-treated mice exhibited reductions in mucus production (Fig. 6).

The relative protein expression levels of iNOS in the lung tissue was significantly increased in the OVA-sensitized/challenged mice, as compared with the normal controls (Fig. 7A and B). However, the TO-treated mice exhibited marked decreases in the protein expression of iNOS, as compared with the OVA-sensitized/challenged mice. The relative protein expression levels of MMP-9 in the lung tissue were similar to that of iNOS. The OVA-sensitized/challenged mice exhibited significantly increased MMP-9 protein expression in the lung tissue, as compared with the normal controls, whereas the TO-treated mice exhibited a marked decrease in the MMP-9 protein expression, relative to the OVA-sensitized/challenged mice.