Rabbit polyclonal anti-P2X4R primary antibody was purchased from Calbiochem (EMD Millipore, Billerica, MA, USA; cat. no. PC376) and used for western blotting. Mouse monoclonal anti-PCNA primary antibody (cat. no. BM0104) and anti-α-SMA primary antibody (cat. no. BM0002) were purchased from Boster Systems, Inc. (Wuhan, China). Mouse monoclonal anti-β-actin primary antibody (cat. no. TA-09) was purchased from ZSGB Biotechnology, Inc. (Beijing, China). Mouse anti-rabbit (cat. no. sc-2357) and rabbit anti-mouse (cat. no. sc-358914) IgG-horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The P2X4R-specific antagonist, 5-BDBD, was obtained from Tocris Bioscience (Bristol, UK). The Masson's trichromatic staining kit and Alcian blue-periodic acid-schiff (AB-PAS) mucosa staining kit were purchased from Fuzhou Maixin Biotechnology, Co., Ltd. (Fuzhou, China). ATP and the P2XR1/2/3/5/7 antagonist, phenyl-isopropyl-amine dopes (PPAD), were all purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were obtained from common commercial sources.

Pathogen-free female BALB/c mice, aged 6–8 weeks and weighing ~18–20 g, were purchased from the Laboratory Animal Research Center (Beijing, China). Mice were maintained under 12 h light/dark cycles at room temperature, with free access to food and water. All animal experiments and manipulation procedures were approved by the Ethics Committee for Animal Use and Care of Harbin Medical University-Daqing (Daqing, China).

A total of 48 BALB/c mice were randomized into the following five groups: Phosphate-buffered saline (PBS) control group (n=8), OVA group (n=10), ATP group (n=10), PPAD group (n=10) and 5-BDBD group (n=10). In the OVA group, the mice were sensitized to OVA (20 µg per injection), absorbed to 2.0 mg per injection of aluminum hydroxide (Tianjin Fuchen Chemical Reagents Factory, Tianjin, China), by intraperitoneal injection on day 1. On days 8, 15 and 22, the mice were sensitized again to OCA (10 µg per injection), absorbed to 1.0 mg per injection of aluminum hydroxide, by intraperitoneal injection. From day 23, the mice were exposed, using inhalation aerosols, of 4% OVA in PBS for 25–30 min, until the onset of bronchial obstruction, daily for 7 days consecutively, according to the methods described by Shen et al (31) and Vanacker et al (32) with modifications. The mice in the ATP, PPAD and 5-BDBD groups were also subjected to OVA sensitization and asthma induction, in the same manner. Intranasal application of 50 µl ATP (1 mM) (33), PPAD (0.1 mM) (34) and 5-BDBD (30 µmol) (35) were performed 3 h prior to each airway allergen challenge in the ATP, PPAD and 5-BDBD groups, respectively. All treatments were administered daily for 5 days consecutively. Mice in the control group were administered with PBS alone (0.5 ml) by injection, and were challenged with PBS (20 ml). All animals were humanely sacrificed within 24 h of the final OVA or PBS exposure.

The lungs were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at a thickness of 5 µm. The sections from each animal were stained with hematoxylin and eosin for inflammation, Masson's trichrome staining for collagen-deposition and Alcian blue/periodic acid-Schiff (AB-PAS) staining for the analysis of goblet cell hyperplasia/mucus production. All staining procedures were performed in accordance with the manufacturer's protocol. Pathological changes in the lung and bronchial tissues were observed and images were captured using a BX-60 microscope (Olympus, Tokyo, Japan). The tissues were scored based on the presence of inflammatory cells, using the following scale: Absent (0), rare (1), mild (2), moderate (3) or severe (4). Scoring was performed by an observer in a blinded-manner. Positively stained areas of collagen and mucus in the lung and bronchial tissues were quantified using high-resolution images of individual vessels by image analysis, using a color-recognition algorithm, in Image Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). The results were expressed as the percentage of collagen area or mucus area, compared to the total measured area.

Samples of BALF were collected 24 h following the final challenge, according to previously described methods. The lungs were lavaged with 1.6 ml (0.8 ml per lavage) of ice-cold PBS via the tracheal cannula. The retrieved volume (~75–80%) of the instilled PBS was then centrifuged at 500 × g at 4°C for 5 min. The resultant pellet was washed twice with PBS and was resuspended in 1 ml PBS. The total numbers of cells were counted using a hemocytometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Smears of the BALF cells were stained with Wright and Giemsa staining fluid (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for differential cell counting. Using a BX-60 Olympus microscope, the numbers of cells were counted by two independent investigators, in a blinded-manner. A total of ~200 cells were counted in each of four randomly selected locations.

The lung tissues were homogenized with cold lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA and 2 mM PMSF. Following ultrasonication on ice for 5 sec using an FA-25 (Fluko Equipment Shanghai Co., Ltd., Shanghai, China) and centrifugation at 12,500 × g at 4°C for 15 min, the supernatant was collected, and the protein concentration was determined using a Bicinchoninic Acid Protein Assay (Pierce Biotechnology, Inc., Rockford, IL, USA) with bovine serum albumin (Biosharp, Hefei, China) as a standard. Each sample, containing 20 µg protein, was separated on 8–10% SDS-PAGE gels and transferred onto nitrocellulose membranes (EMD Millipore). Following blocking with Tris-buffered saline, containing 20 mM Tris, 150 mM NaCl (pH 7.6) and 0.1% Tween 20, with 5% nonfat milk at room temperature, the membranes were incubated overnight at 4°C with specific antibodies against P2X4R (1:400 dilution), α-SMA (1:500 dilution), PCNA (1:500 dilution) or β-actin (1:1,000 dilution). This was followed by incubation for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution). All protein blots were visualized using enhanced chemiluminescence reagents (GE Healthcare Life Sciences). Integrated density values were analyzed using a computerized image analysis system (Fluor Chen 2.0; Olympus Corporation) and were normalized to those of β-actin.

Data were analyzed using SPSS 15.0 software (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard error of the mean. Differences between groups were analyzed using Student's t-test and one-way analysis of variance, followed by Fisher's least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

The present study performed western blotting to compare the protein expression levels of P2X4R in the lung tissues of the treatment groups (Fig. 1A and B). As shown in Fig. 1, OVA-challenge significantly increased the expression of P2X4R, compared with the control mice.

Histological analyses of the tissues of the mice in the OVA group showed more extensive infiltration of inflammatory cells around the bronchioles, blood vessels and alveoli. The airway mucosa showed edema, with bronchiolar wall thickening causing luminal stenosis, suggesting successful establishment of the asthmatic model. In the OVA-challenged mice, treatment with ATP significantly increased OVA-induced inflammation, infiltration, and pathological changes of the bronchi and lung tissues. By contrast, treatment of OVA-challenged mice with PPAD (a P2X1, 2, 3, 5 and 7R antagonist) (36) and 5-BDBD (a P2X4R antagonist) (37) significantly reduced the extent of inflammation and cellular infiltration in the airway. In addition, the pathological changes of the bronchi and lung tissues were milder, compared with the changes found in the asthma group without treatment (Fig. 2A and B).

The total cell numbers (Fig. 3A) and the percentages of eosinophils and lymphocytes (Fig. 3B) in the BALF from the asthmatic mice increased significantly, compared with those in the BALF from the control mice. The increased numbers of inflammatory cells, including eosinophils and lymphocytes, were significantly reduced by the administration of PPAD and 5-BDBD in the asthmatic mice, compared with the numbers found in the untreated asthmatic mice. The changes in the percentages of macrophages showed the opposite results.

Collagen deposition in airway remodeling was assessed using Masson's trichrome staining of lung histological sections. Collagenous matrix was identified by its characteristic blue color. No obvious collagen fibers were present in the lungs of mice in the control group. However, deposition of collagen fibers was increased around the bronchial airways and vessels in the lungs of the OVA-challenged and ATP-treated mice. By contrast, intervention with PPAD and 5-BDBD significantly reduced collagen deposition (Fig. 4A and B).

To assess mucus production and the hyperplasia of goblet cells, lung sections were stained with AB-PAS. In the OVA-challenged mice, bronchial epithelial mucus secretion and goblet cell hyperplasia were clearly observed, as a violet color in the bronchial airways, compared with the control group. The level of goblet cell hyperplasia in the OVA-challenged mice treated with ATP was significantly increased, compared with that in the OVA group. Intervention by PPAD or 5-BDBD significantly reduced goblet cell hyperplasia (Fig. 5A and B).

Western blotting was performed to examine the protein levels of α-SMA (Fig. 6A and B) and PCNA (Fig. 7A and B) in each group. The expression levels of α-SMA in the OVA-challenged mice and ATP-treated mice were significantly lower, compared with those in the control mice. In addition, the mice treated with 5-BDBD showed significantly increased protein expression levels of α-SMA, compared with the mice in the OVA and ATP groups. However, the mice treated with PPAD showed no significant differences in protein expression, compared with the OVA group. The expression level of PCNA in the OVA-challenged mice and the ATP-treated mice were significantly higher than the level in the control mice. In addition, the mice treated with 5-BDBD showed a significant reduction in the protein expression levels of PCNA, compared with the mice in the OVA and ATP groups. Treatment with PPAD had no significant effects.