Asthma model
Female ICR mice (5–6 weeks old) were purchased from Changzhou Cavens Laboratory Animal Ltd. (Changzhou, China). The mice were maintained at 25 ± 2℃ with a 12 h light/dark cycle and fed with a standard rat chow and water ad libitum. All experiments were approved by the Ethical Committee for Animal Experiments of Navy Medical University, and strictly carried out in accordance with the approved guidelines.
Mice were randomly divided into four groups (n = 6 each): (1) control group, (2) 60% H2 group, (3) asthma group, and (4) asthma + 60% H2 group. The mice were sensitized with ovalbumin (OVA) + Al(OH)3 (both from Sigma, St Louis, MO) (100 µg OVA and 3 mg Al(OH)3 in 300 µl PBS) or PBS (300 µl PBS) by subcutaneous injection on day 1, 8 and 15. This was then followed by a nasal drip of 20 µl OVA (5 µg OVA in 20 µl PBS) or PBS (20 µl PBS) on day 22 and 29. To assess the effects of hydrogen gas on asthma, the mice of 60% H2 group and asthma + 60% H2 group were housed in a specific airtight device producing air mixture including 60% H2 and 21% O2 for two weeks (2 hours per day).
Cellular model
16HBE cells were obtained from Puhe Biotechnology Company (Wu Xi, China), and maintained in DMEM high glucose medium containing 10% foetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA). The cellular model of asthma was established by treating 16HBE cells with 10 µg/m house dust mite extract (HDM, Dermatophagoides pteronyssinus, STALLERGENES GREER, Lenoir, NC) for 12 h, and treated with hydrogen in the same way as animal experiments.
Gas production
The mixed gas consisting of H2 and O2 (66.7% : 33.3% v/v) was produced by the AMS-H-01 hydrogen nebulizer (Asclepius, Shanghai, China) used in animal experiments, which was designed to produce hydrogen by electrolyzing water, whereas, the hydrogen incubator (PH-1-A) used in the cell experiments and adjustable three gas chamber (PH-2-A) used for animal experiments were from Puhe Biotechnology Company (Wuxi, China). Thermal trace GC ultra-gas chromatography (Thermo Fisher, MA, USA) was used to monitor the concentration of hydrogen gas in the box.
BALF collecting
Using a 1 ml syringe, 0.5 ml aliquots of 0.9% NaCl solution was injected into the trachea and lungs through the tracheal cannula, the chests of mice were gently massaged and rinsed for approximately 1 min, and then the liquid was withdrawn with the same syringe. The process was repeated 3 times, and the total quantity of BALF was 1.2 ml for each sample. The cellular fraction from BALF was collected by centrifugation at 1500 rpm for 10 min at 4℃ and resuspended in 1 ml saline.
For experiments on the effect of hydorgen on innate immune cell ILC2, BALF cells isolated from normal mice and asthmatic mice were each divided into two groups: (1) control group and (2) 60% H2 group and cultured for 12 h.
Immunohistochemical
The airway tissue were fixed with 4% paraformaldehyde and embedded in paraffin. For immunohistochemistry, deparaffinized sections were incubated with 3% H2O2 for 5 minutes to block endogenous peroxidase activity. After blocking with 10% foetal bovine serum, sections were stained with anti-ZO-1 antibodies (ab190085) or anti-E-cadherin (CST3195S) antibody of interest followed by secondary Abs. Sections were washed three times with PBS buffer. The colour was developed using DAB substrate-chromogen solution (Biocare Medical), and images were obtained under a microscope with 40 magnification (OLYMPUS-IX71).
Flow cytometry
Cells in BALF were collected and prepared to cell suspension (3 × 106 cells/ml). The receptor blocker was incubated at room temperature for 10 min, then the intraprep permeabilization reagent was incubated at 4 ℃ for 20 min. After PBS washing, the corresponding antibodies were incubated at room temperature and away from light for 40 min. Flow cytometry was performed and results were analyzed with CELL Quest software.
ELISA
Serum, BALF supernatant of mice or cell culture medium were collected for ELISA assay of IL-4, IFN-γ, IL-33, IL-25, TSLP, MCP-1, IL-5, IL-9, IL-13 (Elabscience) and ST2 (ab213871, abcam) according to the manufacturers’ recommendation. The data were expressed as cytokine (pg/ml) for each sample.
Immunofluorescence Staining
Cells were fixed with 4% PFA for 15 min and washed with 0.2% Triton X-100 in PBS for 10 min. Then blocked with 1% BSA in PBS, and incubated with monoclonal anti-IL-33 antibody (ab187060, abcam) and anti-ST2 (60112-1-Ig, Proteintech) antibody, followed by DyLight488 goat anti-rabbit IgG secondary antibodies (GAR4882, Multi Sciences) and anti-mouse CY3 secondary antibodies (SA00009, Proteintech) away from light for 1 hr at room temperature before being counterstained with hoechest and mounted.
CCK-8 assay
5000 16HBE cells/well in 100 µl of complete medium were seeded in 96-well plates and culture at 37 °C for 24 hours in a humidified incubator with 5% CO2. Then the cells were treated with 10 µg/ml of HDM and or H2 for 24 h before adding 10 µl of CCK-8 solution to each well and incubating the plate for 3 hours in the incubator. The absorbance at 450 nm was measured using a microplate reader (Molecular Devices, San Jose, CA).
Western blot
Protein quantification was performed. Protein lysate was introduced to SDS-PAGE and subsequently electrotransferred to a polyvinylidene fluoride membrane. Western blotting was carried out as previously reported. The primary antibodies used were listed as follows: anti-IL33 (abcam, ab187060, 1:1000), anti-caspase 3 (abcam, ab90437, 1:1000), anti-cleaved caspase 3 (abcam, ab49822, 1:500), anti-caspase 9 (abcam, ab52298, 1:500), anti-cleaved caspase 9 (abcam, ab2324, 1:1000), anti-p65 (Active Motif, 39159, 1:1000), anti-p-p65 (abcam, ab76307, 1:5000), anti-ST2 (Proteintech, 60112-1-Ig, 1:2000), anti-GAPDH (abcam, ab181602, 1:10000). Secondary antibodies included goat anti-rabbit IgG-HRP (Abcam, Cambridge, MA, USA).
Quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted from airway tissues using RNeasy mini kits (Qiagen, Venlo,Netherlands). Reverse transcription was performed with the SuperScript ® III First-Strand Synthesis System (Life Tech, Shanghai, China) according to the supplier's instructions using 1 g total RNA. Quantitative real-time PCR was performed using the SYBR ® Green PCR Master Mix (Life Tech) on a ABI 7300 (Applied Biosystems, Foster City, CA) with the following program: 95℃for 3 min followed by 40 cycles of 95℃for 30 sec, 58℃ for 15 sec, and 68℃for 30 sec. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd (Shanghai, China). The relative transcription levels were calculated with the 2− ΔΔCt method using gapdh as the internal control.
Luciferase reporter gene assays. The effect of HDM on IL-33 promoter luciferase reporter assays was determined. The IL-33 promotor region was was constructed to the expression vector of pGL3-basic. 16HBE were seeded in 24-well plates (3 × 104 cells/well) and maintained for 24 h. Cells were then serum-starved (0.1% FBS/DMEM) for 24 h, transfected with the pGL3-basic and pRL-TK reporter vector using LipofectAMINE reagent (0.2 µg DNA/well; 1 µl of LipofectAMINE/well in a total volume of 140 µl/well) in serum-free media for 4 h and subsequently treatment with 10 µg/ml HDM or/and 60% H2 for an additional 12 h/24 h. Cells were then treated with test agents at the indicated concentrations for 6 h and luciferase activity measured using a luciferase assay kit and a microplate luminometer (Luminoskan Ascent; LabSystems, Helsinki, Finland). Luciferase activity was normalized to Renilla luciferase.
MiRNA microarray
RNeasy Micro Kit (Qiagen) was used to extract RNA from 16HBE cells following manufacturer’s instructions. The Whole Human miRNA Microarray was a broad view that represents all known miRNAs in the human transcriptome. Sequences were compiled from a broad source survey, and then verified and optimized by alignment to the assembled human transcriptome.
Sample labeling and array hybridization were performed according to the Agilent miRNA Microarray Systemwith miRNA Complete Labeling and Hyb Kit protocol (Agilent Technology). Briefly, total miRNA from each sample was labeled with Cyanine 3-pCp under the action of T4 RNA ligase. The labeled cRNA over the procession of inspissation and desiccation and then redissolved with water. 1 µg of each labeled cRNA was fragmented by adding 11 µl 10 × Blocking Agent and 2.2 µl of 25 × Fragmentation Buffer, then heated at 60 °C for 30 min, and finally 55 µl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 100 µl of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hours at 65 °C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned with using the Agilent Microarray Scanner (part number G2505C).
The chip is washed and scanned using the Agilent Microarray Scanner.Agilent Feature Extraction software is used to collect the chip probe signal value.Chip standardization was carried out with Agilent GeneSpring GX v14.9 software.
Statistics
All quantitative data are expressed as mean ± SD. The software SPSS Statistics 22.0 (SPSS, Chicago, IL) was used to perform the statistical analysis. One-way ANOVA (analysis of variance) was used to verify the differences between groups. P < 0.05 was regarded statistically significant.