A total of 20 female BALB/c mice (specific pathogen-free; aged 5–6 weeks; weight, 16–20 g) were purchased from the Animal Center of Guangdong Province (Guangzhou, China) and fed in a specific pathogen-free grade breeding room (separated into two groups of ten, bred separately and conventionally in cages, at a temperature of 18–22°C, 50–60% humidity and 10–14 h lighting with an air flow of 10–25 cm/min).

The present study was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Shenzhen University (Shenzhen, China). Reagents and chemicals were analytical grade (Sigma-Aldrich, St. Louis, MO, USA) and solvents were HPLC grade (Mallinckrodt Australia Pty Ltd., NSW, Australia). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed using a Mini Protean II apparatus (Bio-Rad Laboratories, Inc., Hercules, California, USA) with 12% gels and a Tris/Tricine buffer system. Two-dimensional PAGE was performed using Immobiline IPG strips (13 cm) with a pH range of 3–10 (GE Healthcare Life Sciences, Shanghai, China). Al(OH)₃ and methacholine were purchased from Sigma-Aldrich. Biotin-labeled goat anti-mouse immunoglobulin IgE was purchased from Novus Biologicals, (Colorado, USA), horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and IgG2a were obtained from eBioscience, Inc. (San Diego, CA, USA). Mouse interleukin [IL; IL-4 (catalog no. 431104) and IL-10 (catalog no. 431414)], and interferon (INF)-γ enzyme-linked immunosorbent assay (ELISA) kits (catalog no. 430801) were obtained from BioLegend, Inc. (San Diego, CA, USA). The vector of pET-28b/Dermatophagoides farinaeI (Derf I) was constructed in the State Key Laboratory of Respiratory Disease for Allergy, Shenzhen University (Shenzhen, China).

Expression of recombinant protein, Derf I was induced using 1 mM isopropyl β-D-1-thiogalactopyranoside at mid-log phase at 37°C and the samples were collected 4 h post-induction. Harvested induced cells were lysed with 10 mg/ml lysozyme in Tris buffer (100 mM NaH₂PO₄ and 10 mM Tris-Cl) and homogenized by sonication. Inclusion bodies were collected by centrifugation at 12,000 × g for 20 min at 4°C and then washed three times with Tris buffer containing 0.5% (v/v) Triton X-100. Inclusion bodies, solubilized in 6 M GuHCl, were purified by Ni-NTA at 4°C. The recombinant protein was electrophoresed (110 V) on 12% (w/v) SDS polyacrylamide gels and its protein concentration was determined using the bicinchoninic acid assay (AppliChem GmbH, Darmstadt, Germany) according to the manufacturer's protocol.

The animal sensitization model was designed for the following experiment. Briefly, the mice were randomly divided into two groups (20 mice per group). The model groups were sensitized intraperitoneally (I.P.) with 50 µg Derf I with 4 mg Al (OH)₃ on days 1, 3 and 7, while the normal group was sensitized, challenged and treated with phosphate-buffered saline (PBS) and 4 mg Al(OH)₃. The model groups were induced with 50 µg/ml Derf I every day for one week, which was substituted with 50 µg/ml PBS in the normal group.

Twenty-four h after the final challenge, AHR was measured using unrestrained whole-body plethysmography with a four-chamber system (Buxco Research Systems, Wilmington, NC, USA) as previously described (25).

The recombinant protein Derf I was diluted with an identical volume of lysis buffer (0.5L) as follows: 9 mol/l Urea, 0.8% IPG buffer (pH, 3–10), 1% dithiothreitol (DTT) and 2% 3-[(3-cholamidopropyl) dimethylammonio] propane-1-sulphonic acid. Subsequently, PAGE and mass analysis were performed as described by Cheng et al (26) and Li et al (27).

Blood samples (1 ml) were obtained from the eyes of the mice, and centrifuged at 4°C and 324 × g for 10 min. The supernatant serum was collected and the IgE, IgG, and IgG2a were subsequently analyzed using an indirect ELISA protocol according to the manufacturer's protocol.

BALF was collected as previously described (28) with slight modifications. Briefly, mice were sacrificed by euthanasia, and BALF was obtained using 1 ml PBS containing protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). Cytospin preparations were prepared and counted using Liu's stain under an optical microscope. Cells were classified as eosinophils according to morphologic and histologic criteria (29).

The splenocyte cells from the mice were cultured in RPMI-1640 medium with 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA) in 24-well plates at a density of 5×10⁴/well, stimulated with the recombinant Derf I protein (200 µg/well), and incubated at 37°C for 72 h. The IL-4, IL-10 and INF-γ levels in the splenocyte culture medium and BALF were detected using an ELISA kit (catalog no. 421701, Biolegend, Inc.).

RT-qPCR was performed to detect the mRNA expression of ATP5b on an Applied Biosystem 7000 Sequence Detection System (Thermo Fisher Scientific, Inc., Waltham, MA, USA), which was performed as described by Ding et al (28) and Zuo et al (30). Five micrograms of total RNA from each sample was reverse transcribed to cDNA using an A3500 RT system (Promega Corporation, Madison, WI, USA). The following forward and reverse primers were used: Forward, AGT TGC TGA GGT CTT CAC GG and reverse, CTT TGC CAC GGC TTC TTC for ATP5b; and forward, TGG CAG AGA TGC GTG GAG A and reverse, GGC AAG TCT TCC GAG TAG TTT T for GAPDH. The PCR conditions were as follows: 5-mindenaturation at 95°C followed by 40 cycles at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec. Amplification of the target gene was monitored as a function of increased SYBR-Green. I (Qiagen GmbH, Hilden, Germany) fluorescence. An analysis threshold was set, and the cycle threshold (Cq) was computed for each sample (31). The comparative difference in gene expression was then determined.

siRNA targeting human ATP5b was delivered into ASMCs using the Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. In addition, a non-targeting siRNA pool (Dharmacon, Inc., Lafayette, CO, USA) was used at the same concentration as a control for the RNA interference assays. After 48 h of transfection, cells were subjected to MTT assays or RNA were collected and analyzed by RT-qPCR.

ASMCs were seeded into96-well culture plates at adensity of 500 cells/well with scramble siRNA-ATP5b and siRNA-ATP5b. After 48 h, the cell culture was removed and 20 µl of MTT was added to each well. The cells were further cultured at 37°C for 4 h, and the cell culture was removed and 150 µl DMSO was added to dissolve crystals completely by pipetting up and down. A microplate reader (SynergyH1; Bio-Tek Instruments, Inc., Winooski, VT, USA) measured the corresponding absorption value at a wavelength of 562 nm indicated cell proliferation as the number of live cells was proportional to the optical density value.

The data are expressed as the mean ± standard deviation. Student's t-test was used for the statistical analysis of interval data and P<0.05 was considered to indicate a statistically significant difference.

In the current study, DerfI as an important antigen of asthma was applied to construct the asthma model. Purified DerfIprotein was obtained (Fig. 1A). AHR changes were assessed by methacholine challenge 24 h after the final allergen challenge. The results (Fig. 1B) demonstrated that AHR in the model groups was significantly greater (P<0.05) than in the normal control group. In addition, the AHR of the mice increased with the increasing concentration of methacholine. Fig. 1C and Table I showed that the total counts of cells and inflammatory cells in the BALF sampled from the model groups were significantly greater than those in the normal group (P<0.05), particularly the eosinophils. As shown in Fig. 1D, lung tissue samples from the model group were damaged by internal hemorrhage and edema. The bronchial and vascular walls became thickened and infiltrated by a marked number of inflammatory cells. In addition, the trachea in the models was filled with markedly more mucus than the normal control group. However, lung tissue structures in the normal group were well defined without discernible damage or edema, and the trachea and blood vessels were not infiltrated peripherally by inflammatory cells. Thus, the asthma model had been successfully induced.

Serum IgE, IgG and IgG2a levels were measured by ELISA to evaluate the asthma model. The results (Fig. 2A) showed that the levels of serum IgEin the asthma model group were significantly higher than those in the normal control group (P<0.05). Furthermore, the concentrations of IL-4, IL-10 and IFN-γ in the splenocyte culture medium supernatant and BALF were detected by ELISA. The asthma model groups (Fig. 2B) exhibited allergen-specific Th2 immune responses, where by IL-4 and IL-10 levels were evidently upregulated compared with the normal control group (P<0.05). However, the IFN-γ level in the asthma model group was significantly lower than that in the normal control group (P<0.05), which enhances inflammatory and immunosuppressive function. These data demonstrated that Derf I protein disturbed the Th1/Th2 balance, which promoted the Th2 immune response and suppressed the Th1 response.

A total of 23 monoisotopic peaks were input into the Mascot search engine to search the Swiss-Prot database (http://www.gpmaw.com/html/swiss-prot.html). The annotation of all the identified proteins is summarized in Table II. The query result showed that protein spot 2606 is the ATP5b protein. In addition, the database query result and mascot score of a representative ATP5b protein are demonstrated in Table III and Fig. 3.

It was further verified by RT-qPCR that ATP5b was expressed in the asthma model mice to a significantly greater extent when compared with the normal control mice (Fig. 4A). In order to confirm the role and function of ATP5b, the experiments were designed to silence ATP5b expressionin ASMCs and detect its effect on cell proliferation (Fig. 4B). The data indicated that silencing of ATP5b inhibited ASMC growth by MTT (Fig. 4C). Thus, overexpression of ATP5b enhances ASMC proliferation.