A total of 36 BALB/c female mice (age, 8–10 weeks; weight, 22±2 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in a controlled environment with 40–50% humidity at 22±1°C under a 12-h light/dark cycle. All mice had access to food and water ad libitum. All animal experiments in the present study were conducted in compliance with the Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of Harbin Medical University (Harbin, China). Asthma was induced using OVA as previously described (15). Briefly, mice were sensitized with 50 µg OVA (Sigma-Aldrich, St. Louis, MO, USA), which was emulsified with 5 mg aluminum hydroxide (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), via intraperitoneal injection on days 0, 7 and 14. From day 15, the mice received an airway challenge with 1% OVA for 30 min, three times a week for 9 weeks.

Following a week of adjustable feeding, the mice were randomly divided into three groups (n=12/group): i) Control group, mice received the same volume of phosphate-buffered saline (PBS) by intraperitoneal injection, and did not undergo airway challenge; ii) OVA group, mice were injected intraperitoneally with 300 µl PBS containing 0.9% ethanol 30 min prior to each airway challenge; iii) vitamin D3 group, mice were intraperitoneally injected with 100 ng 1,25-dihydroxyvitamin D3 (Sigma-Aldrich) dissolved in ethanol, and further diluted to 0.9% by 300 µl PBS, 30 min prior to each airway challenge. A total of 24 h after the last challenge, mice were anesthetized with 3.0 ml/kg 10% chloral hydrate (Sinopharm Chemical Reagent Co., Ltd.) by intraperitoneal injection and lungs were lavaged with 1.5 ml saline three times; the bronchoalveolar lavage fluid (BALF) was collected for further analysis. Subsequently, the mice were sacrificed by 5.0 ml/kg 10% chloral hydrate, and the lungs were harvested for further experimentation.

BALF samples were centrifuged at 156 × g for 10 min at 4°C, the supernatant was stored at −80°C, and the cell pellet was resuspended in 1 ml PBS. The number of total cells was counted using a hemocytometer, and the remaining cells were centrifuged onto slides and stained with Wright-Giemsa in order to count the number of eosinophils.

The levels of total IgE and TGF-β1 in the BALF samples were measured using ELISA kit for Immunoglobulin E and ELISA kit for TGF-β1 (USCN Life Science, Inc., Wuhan, China) according to the manufacturer's protocol. The optical density (450 nm) was measured using a microplate reader (ELx800; Biotek Instruments, Inc., Winooski, VT, USA), and the concentrations of IgE and TGF-β1 was calculated from a standard curve. The serum levels of calcium were measured using a calcium colorimetric assay kit (Sigma-Aldrich) according to the manufacturer's protocol.

For histological analysis, lung tissues were fixed with 10% buffered neutral formalin, embedded in paraffin, cut into 5-µm sections, and stained with hematoxylin and eosin (H&E) using 0.2% hematoxylin for 5 min and 0.35% eosin for 3 min; periodic acid-Schiff (PAS), using 0.5% Schiff staining solution for 15 min; or Masson, using 1% hematoxylin for 6 min and ponceaux-acid fuchsin staining solution (0.7% ponceaux, 0.3% acid fuchsin) for 1 min. For immunohistochemical analysis, the sections were initially incubated with anti-α-SMA mouse monoclonal antibody (1:200; Wuhan Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0002), anti-Nrf2 mouse polyclonal antibody (1:50; Wuhan Boster Biological Technology, Ltd.; cat. no. BA3790) or anti-HO-1 mouse polyclonal antibody (1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-390991) at 4°C overnight, and were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse (1:50; cat. no. A0286) and goat anti-rabbit (1:50; cat. no. A0277) secondary antibodies (Beyotime Institute of Biotechnology, Haimen, China) for 30 min at 37°C. Positive staining was detected with HRP-conjugated streptavidin, visualized with 3,3′-diaminobenzidine and counterstained with hematoxylin. Finally, the sections were mounted, cover-slipped, and examined under a light microscope (DP73; Olympus Corporation, Tokyo, Japan). The extent of lung infiltration as detected by H&E staining, and goblet cell hyperplasia as detected by PAS staining were graded using a semi-quantitative scoring system (17). The collagen area of similar size bronchioles and the α-SMA positive area were analyzed using Image-Pro Plus 4.5 (Media Cybernetics, Inc., Rockville, MD, USA) and are presented as positive area/total area of bronchioles (18) The integrated optical density (IOD) of Nrf2 and HO-1 was also measured by Image-Pro Plus 4.5, and the mean density was calculated as IOD / area.

Total proteins were extracted using radioimmunoprecipitation assay lysis solution (Beyotime Institute of Biotechnology). Nuclear and cytoplasmic protein extracts were prepared using a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology). Protein concentration was determined using the BCA Protein assay kit (Beyotime Institute of Biotechnology) and 40 µg protein were separated by 5, 10 or 14% sodium dodecyl-sulfate polyacrylamide gel electrophoresis, and were transferred onto polyvinylidene fluoride membranes (EMD Millipore, Bedford, MA, USA). After blocking with 5% fat-free milk, the membranes were immunoblotted with primary antibodies as follows: Mouse α-SMA (1:400), rabbit HO-1 (1:200; Santa Cruz Biotechnology, Inc.; cat. no. sc-10789), mouse Nrf2 (1:400), rabbit TGF-β1 (1:200; Santa Cruz Biotechnology, Inc.; cat. no. sc-146), rabbit Smad2 (1:500, BIOSS, Beijing, China; cat. no. bs-0718R), rabbit phosphorylated (p)-Smad2 (1:500; BIOSS; cat. no. bs-5618R), rabbit Smad3 (1:500; BIOSS; cat. no. bs-3484R), rabbit p-Smad3 (1:500; BIOSS; cat. no. bs-5459R), mouse β-actin (1:1,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-47778) and rabbit histone H3 (1:1,000; BIOSS; cat. no. bs-17422R) at 4°C overnight. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit (1:5,000; cat. no. A0216) or goat anti-mouse (1:5,000; cat. no. A0208) IgG (Beyotime Institute of Biotechnology) at 37°C for 45 min. The specific bands were visualized using an enhanced chemiluminescence (ECL) kit (7Sea Biotech, Shanghai, China) according to the manufacturer's protocol. The semi-quantitative analysis was performed using the Gel-Pro Analyzer version 3.0 (Media Cybernetics, Inc.), β-actin or histone H3 was employed as an internal control and the relative protein levels were quantified by the comparison to the control group.

Lung tissues from each group were homogenized in pre-cooled PBS, and were frozen in liquid nitrogen and thawed three times. Following centrifugation at 10,000 × g for 10 min at 4°C, the supernatant was collected, in order to determine the concentrations of hydroxyproline, MDA and GSH, and the activity of SOD. HO-1 activity was evaluated by determining the amount of bilirubin, as previously described (19). All measurements were performed using commercial kits (Hydroxyproline assay kit, Malondialdehyde assay kit, Glutathione Peroxidase assay kit, Total Superoxide Dismutase assay kit, and Total Bilirubin kit) obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China) according to the manufacturer's manual.

To detect the DNA-binding activity of Nrf2, EMSA was performed using a Chemiluminescent EMSA kit (Viagene Biotech, Inc., Tampa, FL, USA). Briefly, 25 µg nuclear protein from each group were combined with biotin-labeled double-stranded oligonucleotide probes containing the specific recognition sequence of Nrf2 (sense strand, 5′-GGGGAACCTGTGCTGAGTCACTGGA-3′; anti-sense strand: 5′-TCCAGTGACTCAGCACAGGTTCCCC-3′). Following separation on a 6.5% polyacrylamide gel, the complex was transferred onto a nylon membrane and was exposed to ultraviolet light for 10 min. Subsequently, the specific bands were detected using HRP-conjugated streptavidin and an ECL kit (7Sea Biotech).

Data are expressed as mean ± standard deviation. Differences between the groups were compared by one-way analysis of variance followed by Bonferroni post-hoc test, or nonparametric Kruskal-Wallis test followed by Dunn's multiple comparisons test using SPSS version 16.0 software for Windows (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of vitamin D3 on asthma, mice were sensitized and challenged with OVA, and the inflammatory cells in the BALF were counted (Fig. 1A and B). As compared with the control group, the number of total cells and eosinophils in the OVA group was significantly increased (P<0.05), whereas these were markedly decreased by vitamin D3 treatment (P<0.05). The concentration of IgE, which is another feature of asthma, was also determined; as shown in Fig. 1C and D, the elevated levels of IgE in the BALF and serum of the OVA group were significantly reduced following vitamin D3 treatment. H&E staining further confirmed that inflammatory cells intensively infiltrated the peribronchial space of OVA-challenged mice, which was accompanied by thickening of the lung mesenchyme; however, these alterations were markedly alleviated by treatment with vitamin D3 (Fig. 1E). These results clearly indicate that vitamin D3 may effectively inhibit OVA-induced airway inflammation.

To determine the effects of vitamin D3 on OVA-induced airway remodeling, the expression of α-SMA, a marker of smooth muscle cells, was detected by immunohistochemistry. As shown in Fig. 2A, OVA challenge resulted in a marked increase in the expression of α-SMA, which was decreased by vitamin D3 treatment. This result was further confirmed by western blotting (Fig. 2B and C). In addition, the amount of hydroxyproline was determined, in order to assess collagen content (Fig. 2D); OVA challenge significantly elevated hydroxyproline levels (P<0.05), which were reversed following treatment with vitamin D3 (P<0.05). Consistent with these results, Masson's staining indicated that OVA-induced collagen deposition in the lungs was markedly reduced by vitamin D3 treatment (Fig. 2E). Furthermore, PAS staining revealed that OVA-induced goblet cell hyperplasia in the airway epithelium was inhibited by vitamin D3 (Fig. 2F). Collectively, these results suggest that vitamin D3 attenuates OVA-induced airway remodeling.

Previous studies have suggested that TGF-β1 is an important mediator of airway disease (20,21); therefore, the activities of TGF-β1 and members of its signal transducing system, Smad2 and Smad3, were determined, in order to investigate whether TGF-β/Smad pathway inhibition is involved in the protective effects of vitamin D3 on OVA-induced airway remodeling. As shown in Fig. 3A–C, OVA challenge resulted in a significant increase in the expression of TGF-β1 in BALF and lung tissues; however, it was markedly reversed by vitamin D3 treatment. Furthermore, the levels of p-Smad2 and p-Smad3 in the lungs were markedly upregulated by OVA challenge and were further dampened by vitamin D3 treatment (Fig. 3D–F). Overall, these data indicate that the TGF-β/Smad pathway is associated with vitamin D3-mediated protection of OVA-induced airway remodeling.

To evaluate the effects of vitamin D3 on OVA-induced oxidative stress, MDA and GSH content, and SOD activity were detected in the lungs using commercial kits. As shown in Fig. 4, MDA content was significantly increased in the OVA group (P<0.05), which was accompanied by a marked decrease in SOD activity and GSH content (P<0.05); however, these effects were significantly attenuated by vitamin D3 treatment.

The Nrf2/HO-1 signaling pathway has a pivotal role in antioxidant reactions (22,23); therefore, to investigate whether it is associated with the protective effects of vitamin D3 on OVA-induced oxidative damage to the lungs, the expression and activity of Nrf2 and HO-1 were examined. As expected, immunohistochemistry demonstrated that the expression of Nrf2 was significantly increased in the bronchial epithelial cells of the OVA group (Fig. 5A; P<0.01), and was further upregulated in the vitamin D3 group (P<0.01). In addition, nuclear Nrf2 levels were significantly higher in the OVA group compared with the control group (Fig. 5B; P<0.01), and were further upregulated following vitamin D3 treatment (P<0.01). Similar results were observed by EMSA (Fig. 5C); Nrf2 binding activity was elevated in the OVA group, and it was intensified by vitamin D3 treatment. In accordance with Nrf2, HO-1 levels were also markedly upregulated in airway epithelial cells from the OVA group, the levels of which were further enhanced by vitamin D3 treatment (Fig. 5D and E). Furthermore, OVA challenge resulted in a marked increase in HO-1 activity, which was markedly enhanced by vitamin D3 treatment (Fig. 5F). These data indicate that vitamin D3 protects airways from OVA-induced oxidative injury, at least partially via activation of the Nrf2/HO-1 signaling pathway.

A previous study demonstrated that high doses of vitamin D3 may result in a surfeit of calcium in the blood (24); therefore, the serum levels of calcium were measured to evaluate the effects of vitamin D3 on blood calcium levels. The results demonstrated that calcium concentration in the vitamin D3 group was significantly increased (Fig. 6; P<0.01). These results suggest that the asthma protective effects of vitamin D3 are accompanied by hypercalcemia.