Animals
All animal experiments were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. All animal care and experimental procedures used in the present study were approved by the Ethics committee of Beijing University of Traditional Chinese Medicine. Healthy wild-type (WT) C57BL/6 mice (6–8 weeks old, body weight 16–20 g) were obtained from Beijing University of Traditional Chinese Medicine. IL-3 gene disrupted (IL-3−/−) mice on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME). All of our current studies were performed using male mice. The animals were housed in individually ventilated cages under a 12 h light/dark cycle. Before the experiment, mice were habituated to the environment for at least 1 week. Standard chow and water were provided ad libitum. All procedures were performed as humanely as possible to minimize animal suffering.
Experimental protocol
Mice were assigned to four groups (
n = 8): sham+WT mice, sham+IL-3
−/− mice, hyperoxia+WT mice, and hyperoxia+IL-3
−/− mice. In brief, the mice were exposed to 100% O
2 in a specially constructed plexiglas chamber to induce hyperoxic ALI [
16]. Mice in sham group were exposed in room air. Seventy-two hours after hyperoxia or room air challenge, mice were sacrificed after anesthesia by pentobarbitone (50 mg/kg intraperitoneal injection) and were exsanguinated through the vena cava. Then, lung tissue sampling (for lung histology, and lung wet to dry weight ratio) was collected. Bronchoalveolar lavage fluids (BALF) and pulmonary tissue samples (for preparation of lung tissue homogenates) were collected in separate experiments.
Lung water content
To quantify the magnitude of pulmonary edema, we evaluated the dry to wet (D/W) ratio of the lung. The left main bronchi were clamped, and the wet left lung was harvested. They were then placed in an oven for 48 h at 80 °C. Lung water content was calculated as (1-D/W) × 100%.
Collection of BALF
The trachea was cannulated, and the lung was lavaged with 0.5 mL PBS for six times. The recovery rate of BALF was > 90% in all samples. Collected BALF was centrifuged at 1,200 rpm for 3 min. The supernatant was collected for further study.
Enzyme linked immunosorbent assay (ELISA)
The concentrations of tumor necrosis factor (TNF)-α, IL-6, and IL-3 were measured by ELISA according to the manufacturer’s instructions (R&D Systems Inc., Minneapolis, MN, USA). The DNA-binding activity of NF-κB p65 was determined using an ELISA NF-κB p65 transcription factor assay kit according to the manufacturer’s instructions (Chemicon, Temecula, CA, USA).
Western blotting analysis
Cytoplasmic and nuclear proteins were extracted from frozen lung tissue with the Nuclear/Cytosol Extraction kit (BioVision, Inc., Mountain View, CA, USA) according to the manufacturer’s instructions. Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). 50 μg of total protein were subjected to SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk at room temperature for 3 h, incubated with primary antibodies (Anti-NF-κB p65 (sc-7151) and phosphorylated IκBα antibodies (sc-7977), diluted 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA; Anti-phosphorylated IKKα/IKKβ antibody (2078), diluted 1:500, Cell Signaling, Boston, MA, USA; Anti-IL-3 antibody (AF-403-NA), diluted 1:200, R&D Systems Inc., Minneapolis, MN, USA; Anti-CD123 antibody (106002), diluted 1:200, Biolegend, San Diego, CA, USA) at 4 °C overnight. β-actin (3700) and Lamin B (12586) (Cell Signaling, Boston, MA, USA) were used as an internal control for cytoplasmic and nuclear protein, respectively. On the next day, membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling, Boston, MA, USA) at 37 °C for 1 h. Protein bands on the membrane were visualized with ECL Kit (Biovision, Milpitas, CA, USA) using FluorChem FC3 system (ProteinSimple, San Jose, CA, USA). Results were presented as densitometric ratio between the protein of interest and the loading control.
Statistical analysis
All data were analyzed with GraphPad Prism 6.0 (GraphPad Software, CA, USA) and were presented as mean ± SEM. Two-way ANOVA with Bonferroni’s multiple-comparisons test was used for multiple group analysis. Histopathologic scores were compared using the Mann-Whitney U test. The survival rate was estimated by the Kaplan-Meier method and compared by log-rank test. P < 0.05 was accepted as statistically significant.