Female C57BL/6 mice (4-6 weeks old) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). AE2 and cubic cell-specific Brg1-knockout (SFTPC-Cre/Brgfl/fl) mice were generated by hybridizing SFTPC-rtTA/(TetO)7 transgenic mice (The Jackson Laboratory, CA, USA) with Brg1loxP/loxP mice (kindly provided by Prof. Yujun Shi, West China hospital of Sichuan University). Brg1 deletion in SFTPC-Cre/Brg1fl/fl mice was achieved by administering doxycycline (1 mg/mL) in drinking water containing 0.4% sucrose. The various control mice were Brg1fl/fl mice (no Cre) that received doxycycline. Experimental mice were housed under specific pathogen-free conditions and subjected to a 12 h/12 h dark/light cycle. The study was approved by the ethics committee of Chongqing Medical University.
2.2. Grouping and administration
Twenty female C57BL/6 mice were divided into control and asthma groups. Mice in the asthma group underwent house dust mite (HDM) challenge according to previous studies . Briefly, mice received either 20 μg HDM (Greer, Los Angeles, CA, USA) dissolved in 30 μL normal saline (NS) (asthma group) or the same volume of NS (control group) by nasal inhalation on days 0, 14, 21, 23, 25, 27, and 29. A total of twenty SFTPC-Cre/Brgfl/fl mice were divided into control/Brgfl/fl and asthma/Brgfl/fl groups. The modeling method of the control and asthma groups of SFTPC-Cre/Brgfl/fl mice was the same as that of wild-type mice. All mice were sacrificed at 24 h after the last challenge and then euthanized by intraperitoneal injection of 4% pentobarbital (0.5 mg/kg).
2.3. Measurement of airway hyper-responsiveness (AHR)
AHR was determined using an invasive pulmonary function instrument (EMKA Technologies, Paris, France) within 24 h after the final HDM challenge. Mice were anesthetized with 2% pentobarbital sodium, intubated, and placed in whole-body plethysmography chambers. Airway resistance and compliance were measured every second. Baseline and changes in airway resistance were determined using NS and methacholine (3.125, 6.25, 12.5, 25, or 50 mg/mL) respectively, which were nebulized at a volume of 20μL . Lung resistance at each concentration was calculated.
2.4. Histological and immunohistochemical assessment of lung tissues
The left lung tissues were fixed in 4% formalin buffer and embedded in paraffin. The paraffin blocks were then serially sectioned into 4-μm-thick slices and subjected to immunohistochemical (IHC) staining. Antibodies against MUC5AC (1:300; Abcam, Massachusetts, USA) were used for IHC staining. Tissues or cells that were stained brown were recorded as being positive for MUC5AC. The intensity of IHC staining was quantified using Image-Pro Plus 6.0. Regarding the histochemical analysis, paraffin blocks were serially sectioned into 4-μm-thick slices and subjected to hematoxylin/eosin (H&E) or periodic acid-schiff (PAS) (Leagene Biotechnology Co. Ltd, Beijing, China) staining. Three individual areas per section and lung tissues per group were evaluated for inflammatory cell infiltration.
2.5.Broncho alveolar lavage fluid (BALF) analysis and cell counting
The right lung of each animal was washed three times with 0.5 mL phosphate-buffered saline (PBS) in order to collect BALF. Approximately 1.3 mL of the instilled fluid was consistently recovered. All the cells in BALF were collected by centrifugation and treated with red blood lysis buffer, after which they were counted by microscopy using a cell counter. The remaining inflammatory cells were smeared on slides, dried, and stained using Wright-Giemsa stain (Jiancheng Techno Co, Nanjing, China), according to the manufacturer’s instructions, to determine differential cell counts in accordance with conventional morphological criteria. At least 200 cells per slide were evaluated to determine the differential cell counts.
2.6. Cell culture and Treatments
Human bronchial epithelial cells (16HBE) were obtained from the American Type Culture Collection (ATCC, USA) and cultured in Dulbecco’s phosphate-buffered saline medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco, USA). The cells were cultured at 37°C in a 5% CO2 atmosphere, and the growth status of the cells was evaluated under a microscope. Cells were grown to 85-90% confluence, passaged, and subjected to trypsin digestion. Short hairpin RNAs (shRNAs) against Brg1 (Brg1-shRNA), as well as a negative control shRNA (NC-shRNA),were designed and cloned into pGMLV-SC5 RNAi backbone adenoviral vectors (Genomedtech, Shanghai, China). 16HBE cells were then infected with the Brg1-shRNA adenovirus vectors and screened for the stable expression of green fluorescent protein.
2.7. Real-time RT-PCR
Total RNA from 16HBE cells and lung tissues was purified, and cDNA synthesis was performed using a PrimeScript RT Reagent Kit (Takara, Otsu, Japan). PCR reactions were performed using Real MasterMix (SYBR Green, Tiangen, Beijing, China). The primer sequences of Brg1 were forward 5-GACCAGCACTCCCAAGGTTAC-3 and reverse 5-CTGGCCCGGAAGACATCTG-3. The primer sequences for MUC5AC were forward 5-CAGCAGATCATCCGTCAGCAA-3 and reverse 5-ATCGCAGCGCAGAGTCACA-3. The primer sequences for GAPDH were forward 5-CAGCGACACCCACTCCTCCACCTT-3 and reverse 5-CATGAGGTCCACCACCCTGTTGCT-3. The primer sequences for the β-actin were forward 5-CTCCATCCTGGCCTCGCTGT-3 and reverse 5-GCTGTCACCTTCACCGTTCC-3.
2.8. Immunofluorescence detection
The expression of MUC5AC in 16HBE cells was determined using immunofluorescence staining. Three groups of cells (16HBE, Brg1-sc, Brg1-sh) were made into cell slides, and the slides were removed 24 h later and fixed with 4% paraformaldehyde. The slides were stained with primary antibodies, rabbit anti-Brg1 (1:200, Abcam, UK) for 12 h at 4°C. The sections were washed three times with PBS and stained for 30 min at room temperature with donkey anti-rabbit Cy5 secondary antibodies (1:500; Invitrogen, USA). Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole) (Invitrogen, USA), and fluorescence of the samples was captured using a confocal laser scanning microscope (A1þR, Nikon, Tokyo, Japan).
2.9. Western blot and Co-immunoprecipitation (Co-IP) assay
Total protein from the right lung tissue and 16HBE cells was isolated using a total protein extraction kit (KeyGen BioTECH, China). Protein concentration was determined using the bicinchoninic acid (BCA) assay following the standard protocol. Protein samples (30 µg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene ﬂuoride (PVDF) membranes. Membranes were then blocked and incubated overnight with primary antibodies and subsequently incubated with horseradish peroxidase-linked goat anti-rabbit IgG secondary antibody (1:5000; Proteintech, China).The primary antibodies included mouse anti-Brg1 (1:300; Abcam, USA), rabbit anti-MUC5AC (1:500, Proteintech, China), rabbit anti-phospho-JAK1/2 (1:500; CST, USA), rabbit anti-JAK1/2 (1:500, Proteintech, China), rabbit anti-phospho-STAT6 (1:500; CST, USA), rabbit anti-STAT6 (1:500, Proteintech, China), and rabbit anti-β-actin (1:1000; Proteintech, China). Immunoreactivity was visualized using an enhanced chemiluminescence (ECL) kit (Thermo Fisher, USA). Band intensities were quantified using Quantity One software (version 4.6.2; Bio-Rad, Hercules, CA, USA) and normalized to β-actin. The Co-IP assay  was performed using Protein A/G agarose (Beyotime, China), following the manufacturer's instructions, and normal rabbit anti-IgG (Abcam, UK) was used as a control antibody. Harvested samples containing input (protein pretreated with nothing), IgG (protein pre-treated with A/G agarose and rabbit anti-IgG), anti-JAK1/2 (protein pre-treated with A/G agarose and rabbit anti-PI3K), and anti-Brg1 (protein pre-treated with A/G agarose and rabbit anti-Brg1) were analyzed by western blotting.
2.10. Chromatin Immunoprecipitation（CHIP）
16HBE and Brg1 knockdown 16HBE cells (Brg1-sh) were cultured for 48 h. One plate of cells (10 cm plate) was removed for formaldehyde cross-linking and ultrasonic disruption. We chose the CHIP kit from the American company Milipore, and all the experimental steps were performed in accordance with the kit's instructions. The concentration of Brg1 antibody used in CHIP is 1:20, and the antibody was purchased from Zen-bio Biotechnology Co., Ltd., Chengdu, China. The ultrasonic fragmentation product was removed, and the ultrasonic effect was tested by electrophoresis. The next day, the immune complex was precipitated and washed. After de-crosslinking, DNA fragments were recovered. The final sample was dissolved in 30 uL Elution buffer. Finally, the recovered DNA was subjected to q-PCR analysis. The primer sequences of JAK1 were forward 5-GGCGTCGAAGCAAACTGTTT-3 and reverse 5-TACCGTTGTGGGCTACGTTC-3. The primer sequences of JAK2 were forward 5-GACAGACCTGAAGAGCAGCA-3 and reverse 5-CTGGCACCAGATCGTGTAGG-3.
2.11. Statistical analyses
Data are expressed as the mean ± standard error of the mean. GraphPad Prism software (version 5.0; GraphPad, San Diego, CA, USA) was used for the statistical analyses. Two-way analysis of variance was conducted to determine statistically significant differences in the tested variables among the different groups. If an overall test was significant, Tukey’s test was used for specific comparisons between groups. Statistical significance was set at p < 0.05. All experiments were repeated at least thrice with consistent outcomes.