Forty-two specific pathogen-free (SPF), inbred, female BALB/c mice (age, seven weeks; weight, 20–30 g) were obtained from the in-house animal production facility of the Yanbian University Health Science Center (Yanji, China). The mice were maintained in an animal facility under a 12-h light/dark cycle at 22°C in standard laboratory conditions for one week prior to the experiments. Water and a standard diet were provided ad libitum. The experiments were performed in compliance with the guidelines approved by the Institutional Animal Care and Use Committee of the Yanbian University School of Medical Sciences (Yanji, China). Mice were immunized intraperitoneally with 10 µg ovalbumin (OVA; chicken egg albumin; Sigma-Aldrich, St. Louis, MO, USA) with 1.0 mg aluminum hydroxide adjuvant (Imject^® Alum; Pierce, Rockford, IL, USA). A booster injection of 10 µg OVA plus 1.0 mg aluminum hydroxide adjuvant was given 10 days after the initial OVA/adjuvant treatment. From days 17–19, the immunized mice were challenged by exposure to an aerosol of 1% OVA in phosphate-buffered saline (PBS) for 20 min. Bronchoprovocation was performed in a vented plastic chamber (18×14×8 cm) that was adapted for mice. Aerosol particles of 3–5 mm in diameter were created using an ultrasonic nebulizer (NE-U12; Omron, Tokyo, Japan), directed into the plastic chamber and vented to a fume hood. Each group consisted of seven animals. The saline-treated mice that served as controls were exposed to aerosolized saline. G-Rh2 (98% purity) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and dissolved in 0.1% dimethyl sulfoxide (DMSO). The animals received G-Rh2 (50 or 100 mg/kg) or dexamethasone (DXM; 0.5 mg/kg) by oral gavage at 24-h intervals, beginning 1 h prior to the first provocation (challenge), on days 17–19.

Immediately following the assessment of airway responsiveness, mice were anesthetized by intra-peritoneal injection of pentobarbital (50 mg/kg; Wuhan Boster Biological Technology, Ltd., Wuhan, China) and the trachea was cannulated while gently massaging the thorax, after which the lungs were lavaged with 0.7 ml PBS. BAL fluid samples were collected and the number of cells in a 0.05-ml aliquot was counted using a hemocytometer. The remaining samples were centrifuged at 200 x g for 10 min at 4°C (model 5424R centrifuge, Eppendorf-Netheler, Hamburg, Germany) and the supernatants were stored at −70°C for analyses of IL-4, IL-5, IL-13 and IFN-γ levels. Cell pellets were re-suspended in PBS and cytospin preparations (Cytospin 3; Shandon Life Sciences, Astmor, UK) of the BAL cells were stained with Diff-Quik solution (International Reagents, Kobe, Japan). Two independent, blinded investigators counted the cells using a microscope. Approximately 400 cells were counted in each of four different random fields of view (CX31 microscope; Olympus Corporation, Tokyo, Japan). Inter-investigator variation was <5%. The results were expressed as the mean value of the cell counts determined by the two investigators.

IL-4, IL-5, IL-13 and IFN-γ levels in BAL and IgE in serum were determined using ELISA kits for mice. The IL-4 kit was obtained from Invitrogen Life Technologies (Carlsbad, CA, USA) and IL-5, IL-13, IFN-γ and IgE kits were obtained from R&D Systems Inc. (Minneapolis, MN, USA). The enzyme immunoassays were performed according to the manufacturer's instructions.

Total IgE and OVA-specific IgE were measured by ELISA. Briefly, 96-well microtiter plates were coated overnight with an isotype-specific coating (total IgE) and 10 mg/ml OVA in PBS-Tween 20 (OVA-specific IgE). After washing and blocking of the plates, the samples were incubated for 2 h. Subsequently, the plates were washed and horseradish peroxidase-conjugated goat anti-mouse total IgE and OVA-specific IgE were added. After washing the samples four times in PBS-Tween-20 solution, 200 µl o-phenylenediamine dihydro-chloride (Sigma-Aldrich) was added to each well. The plates were incubated for 10 min in the dark and absorbance was determined at 450 nm using a Bio-Rad 680 microplate ELISA reader (Bio-Rad Laboratories, Hercules, CA, USA). Total IgE and OVA-specific IgE concentrations were calculated from a standard curve that was generated using 250 ng/ml recombinant IgE.

After BAL preparation, the lungs were resected, fixed with 4% paraformaldehyde and embedded in paraffin. Specimens were cut into 4-µm sections using a Leica model 2165 rotary microtome (Leica Microsystems, Wetzlar, Germany). The microsections were stained with hematoxylin and eosin (Richard-Allan Scientific, Kalamazoo, MI, USA) and examined under the CX31 microscope (magnification, ×100).

Freshly isolated lung tissue samples were washed and lysed in two volumes of lysis buffer A (50 mM Tris-HCl, pH 7.5, 1 M EDTA, 10% glycerol, 0.5 mM dithiothreitol, 5 mM MgCl₂, 1 mM phenylmethanesulfonylfluoride (PMSF) and protease inhibitor cocktails) for 5 min at 4°C. The resulting suspension was centrifuged at 1,000 xg for 15 min at 4°C. The supernatant fraction was incubated on ice for 10 min and centrifuged at 100,000 x g for 1 h at 4°C to obtain cytosolic protein extracts. The pelleted nuclei were re-suspended in buffer B (1.3 M sucrose, 1.0 mM MgCl₂, 10 mM potassium phosphate buffer, pH 6.8) and centrifuged at 1,000 xg for 15 min. The pellets were suspended in buffer B with a final sucrose concentration of 2.2 M and centrifuged at 100,000 xg for 1 h. The resulting nuclear pellets were washed once with a solution containing 0.25 M sucrose, 0.5 mM MgCl₂ and 20 mM Tris-HCl (pH 7.2), and centrifuged at 1,000 xg for 10 min. The nuclear pellets were solubilized with a solution of 50 mM Tris-HCl (pH 7.2), 0.3 M sucrose, 150 mM NaCl, 2 mM EDTA, 20% glycerol, 2% Triton X-100, 2 mM PMSF and protease inhibitor cocktails, and the resulting mixture was kept on ice for 1 h with gentle stirring, after which it was centrifuged at 12,000 x g for 30 min. The resulting soluble nuclear protein supernatant samples were used for western blot analysis.

Lung tissue samples were homogenized in the presence of protease inhibitors and protein concentrations were determined using the Bradford reagent (Bio-Rad Laboratories). Samples of protein from the lung homogenates (30 µg per lane) were subjected to 12% SDS-PAGE and transferred onto nitrocellulose membranes. Western blot analysis was performed using the following antibodies at a dilution of 1:1,000: Rabbit anti-mouse polyclonal antibodies against IL-5 (cat. no. sc-7887), p38 MAPK (cat. no. sc-535), phospho-p38 MAPK (cat. no. sc-101759), poly(adenosinediphosphate) ribose polymerase (PARP) (cat. no. sc-25780), NF-κB p65 (cat. no. sc-109), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α) (cat. no. sc-847) and β-actin (cat. no. sc-130656) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) as well as IL-13 (cat. no. AF-413-NA; R&D Systems) or IL-4 (cat. no. AAM36; Serotec, Oxford, UK), or the goat anti-mouse polyclonal antibody IFN-γ (cat. no. sc-9344; Calbiochem, San Diego, CA, USA). The binding of all the antibodies was detected using an ECL detection system (Amersham, Arlington Heights, IL, USA) according to the manufacturer's instructions.

Airway responsiveness was measured two days after the last OVA challenge according to the method of Choi et al (25). Conscious unrestrained mice were placed in a barometric plethysmographic chamber (OCP3000; All Medicus, Seoul, Korea), and baseline readings were collected and averaged for 3 min. Aerosolized methacholine (Mch; All Medicus) at increasing concentrations (from 2.5 to 50 mg/ml) was nebulized through an inlet of the main chamber for 3 min, and readings were taken and averaged for 3 min after each nebulization. The bronchopulmonary resistance was determined by calculating enhanced pauses (Penh) according to the manufacturer's protocol with the following equation: Penh = ( T e / R T − 1 ) × ( P E F / P I F )where Te was the expiratory time, RT was the relaxation time, PEF was the peak expiratory flow and PIF was the peak inspiratory flow. The results are expressed as the percentage increase in Penh over the baseline value after PBS challenge (taken as 100%) following a challenge performed with each concentration of Mch.

Values are expressed as the mean ± standard error of the mean. Statistical evaluation of the data was performed using analysis of variance, followed by Dunnett's post-hoc test. SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform analyses and P<0.05 was considered to indicate a statistically significant difference.

In BAL fluid, the total cell number and the numbers of eosinophils, lymphocytes and neutrophils were significantly increased 24 h after OVA inhalation in comparison with those measured 24 h after saline inhalation (Fig. 1A). Of note, these effects of OVA inhalation were significantly reduced by administration of G-Rh2 or positive control DXM.

Histological analyses revealed widespread perivascular and peribronchiolar inflammatory cell infiltrates, which are typical pathological features of asthma, in OVA-challenged mice, but these features were not found in control mice. OVA-exposed mice treated with G-Rh2 or DXM (Fig. 1B) showed significantly reduced inflammatory-cell infiltration in the peribronchiolar and perivascular regions in comparison with that in mice exposed to OVA only. These results suggested that G-Rh2 inhibits inflammatory-cell infiltration and attenuates antigen-induced airway inflammation.

Western blot analysis revealed that IL-4, IL-5 and IL-13 protein levels in lung tissue were significantly increased 24 h after OVA inhalation in comparison with levels measured 24 h after saline inhalation. The OVA-induced increases in IL-4, IL-5 and IL-13 protein levels were significantly inhibited by administration of G-Rh2 or DXM (Fig. 2A). Consistent with these results, ELISAs revealed that protein levels of IL-4, IL-5 and IL-13 in BAL fluid were also significantly increased 24 h after OVA inhalation in comparison with levels measured 24 h after saline inhalation (Fig. 2B), and these changes in protein abundance were also significantly inhibited by administration of G-Rh2 or DXM.

To determine the effect of G-Rh2 on the Th1 response, IFN-γ abundance in lung tissue was assessed using western blot analysis. IFN-γ abundance in OVA-challenged mice was reduced in comparison with that in control mice (Fig. 3A). However, administration of G-Rh2 or DXM inhibited OVA-induced reductions in IFN-γ abundance. Similarly, ELISAs of BAL fluid showed that IFN-γ protein abundance was significantly reduced in OVA-challenged mice in comparison with that of control mice (Fig. 3A), and pre-treatment with G-Rh2 or DXM inhibited this change in expression.

The total IgE and OVA-specific IgE levels in each experimental group were determined by ELISA 24 h after the final OVA challenge. Total IgE and OVA-specific IgE levels in serum were significantly increased 24 h after OVA inhalation in comparison with levels measured 24 h after saline inhalation (Fig. 4). In comparison with the OVA-challenged group, increased total IgE and OVA-specific IgE levels in serum were significantly reduced in the groups treated with G-Rh2 or DXM.

Western blot analysis revealed that nuclear NF-κB p65 abundance in the lung was increased 24 h after OVA inhalation in comparison with that in the control mice, and this effect was inhibited by administration of G-Rh2 or DXM. By contrast, cytosolic NF-κB p65 abundance was reduced 24 h after OVA inhalation in comparison with that of the control mice (Fig. 5A and B), and this effect was also inhibited by administration of G-Rh2 or DXM. The effect of G-Rh2 on OVA-induced phosphorylation and degradation of IκB-α was studied to examine the molecular mechanisms by which G-Rh2 inhibits NF-κB transcriptional activity. G-Rh2 significantly blocked OVA-induced phosphorylation and degradation of IκB-α (Fig. 6A and B). These results indicated that G-Rh2 prevents NF-κB translocation in OVA-challenged mice by blocking phosphorylation and degradation of IκB-α.

Western blot analysis was used to determine the phosphorylation/activation status of p38 MAPK, which is an upstream mediator of NF-κB activation. Phospho-p38 protein abundance in the lung was increased 24 h after OVA inhalation in comparison with that of the control mice, and this effect was inhibited by administration of G-Rh2 or DXM (Fig. 7A and B). However, no significant changes in p38 MAPK protein abundance were observed in any group.

The present study investigated the effect of G-Rh2 on the development of AHR in mice. Airway responsiveness (determined by calculating Penh) induced by methacholine inhalation was increased in OVA-challenged mice in comparison with that in control mice, and this effect was inhibited by administration of G-Rh2 or DXM, as shown in Fig. 8.