Animals and treatments
Female C57BL/6 mice (6 to 8 weeks old) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). The 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. A total of 40 mice were divided into five groups, including control group, asthma group, Dex group, PNS-R1 group and Dex + PNS-R1 group (n = 8 in each group). To induce asthma, mice were sensitized to 20 μg of house dust mite (HDM; Greer, Los Angeles, CA, USA) in 30 μl normal saline (NS) via nasal inhalation on days 0, 14, 21, 23, 25, 27 and 29 . The control mice were similarly treated with NS. On the last 3 days, mice in the PNS-R1 group were administered 5 mg/kg PNS-R1 (ip) (Macklin, Shanghai, China) [14, 15] at 30 min before HDM inhalation. Mice in the Dex group were administered with 5 mg/kg Dex (ip) (Axxora, Farmingdale, NY, USA) at 30 min before HDM inhalation . While mice in the PNS-R1 + Dex group were administered 5 mg/kg PNS-R1 (ip) and 5 mg/kg Dex (ip) at 30 min before HDM inhalation. All mice were sacrificed at 24 h after the last HDM inhalation and then euthanized by intraperitoneal injection of 4% pentobarbital (0.5 mg/kg).
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 5% CO2 atmosphere, and the growth status of cells was evaluated under a microscope. Cells were grown to 85% to 90% confluence, cells passaged, and subjected to trypsin digestion. Cells were divided into control group (16HBE), Dex group (Dex), PNS-R1 group (PNS-R1) and Dex + PNS-R1 group (Dex + PNS-R1). Cells in Dex group were treated by Dex (5 μM)  and PNS-R1 group were treated by PNS-R1 (2 μM)  for 24 h. The Dex + PNS-R1 group cells were treated by both Dex (5 μM) and PNS-R1 (2 μM).
Immunohistochemical (IHC) staining
Left lung tissues of mice 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 IHC staining. The sections were deparaffinized in xylene for 25 min and rehydrated in ethanol for 5 min. The sections were incubated in 3% H2O2 for 10 min; 10 mM sodium citrate buffer was used for antigen retrieval in a microwave oven for 3 min at high power and 15 min at low power. The sections were then blocked in goat serum (Wuhan Boster Biological Technology, Wuhan, China) for 30 min at room temperature. Rabbit antibody against caspase3 (1:500; Proteintech, Wuhan, China) was used for IHC staining overnight at 4 °C, followed by a 30 min incubation with a secondary goat anti-rabbit antibody at 37 °C. The sections were washed in PBS and incubated with 3,3-diami-nobenzidine (DAB, Zhongshan Golden Bridge, Beijing, China) for 3 min. Slides were counterstained with hematoxylin before microscopic analysis.
Microscope and image analysis
The caspase3 expression in lung tissues was subjected to microscopic analysis. Briefly, after IHC staining, if a cell or tissue was stained from light yellow to brown, it would be recorded as positive immunostaining. The areas from bronchial epithelial cells were selected for analysis. The intensity of the staining signal was measured and documented using the Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc. Silver Spring, MD USA). The mean densitometry of the digital image (×200) is designated as representative integral optical density (IOD)/area staining intensity (indicating average optical density analysis). The signal density of tissue areas from three randomly selected visions of slice 3–6 different animal tissues in each group were counted blindly and subjected for statistical analysis.
Western blot analysis
The proteins in each right lung tissue and 16HBE cells were isolated using a total protein extraction kit (KeyGen BioTECH, Jiangsu, China). Protein concentration was determined by conducting a bicinchoninic acid assay following standard techniques. Protein lysis samples (30 µg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Afterwards the samples were transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were then blocked and incubated with the primary antibody 4 °C overnight, and subsequently incubated with the secondary goat anti-rabbit antibody (1:2000; ProteinTech, Wuhan, China). The primary antibodies includes rabbit anti-caspase3 (1:500; ProteinTech, China), rabbit anti-caspase9 (1:500; ProteinTech, China), rabbit anti-Bcl2 (1:200; Abcam, UK), rabbit anti-Bax (1:200; Abcam, UK), rabbit anti-Cyt-c (1:500; CST, USA), rabbit anti-Apaf-1 (1:500; CST, USA), rabbit anti-cleaved-caspase9 (1:500; ProteinTech, China), rabbit anti-cleaved-caspase3 (1:500; ProteinTech, China) and rabbit anti-β-actin antibodies (1:1000; ProteinTech, Wuhan, China). Band intensities were quantified using Quantity One software (version 4.6.2; Bio-Rad, Hercules, CA, USA) and normalized relative to that of the inner control.
To detect cell apoptosis, 16HBE cells were seeded in six-well plates (Corning, NY, USA) and divided into the following four treatment groups: PNS-R1 + Dex group, PNS-R1 group, Dex group and control group. Cells were treated with Dex (5 μM) and PNS-R1 (2 μM) for 24 h. Apoptosis was measured by flow cytometry using the Annexin V-PI apoptosis-detection kit (KeyGen BioTech, China) according to the manufacturer’s instructions. To detect the cell mitochondrial membrane potential (Δψm), we also used the JC-1 apoptosis-detection kit (KeyGen BioTech, China). The percentage of apoptotic cells was calculated using the FASC Calibur MT flow cytometer (BD Bioscience, NJ, USA).
TUNEL assay was performed to detect the apoptotic tracheal epithelial cells in the lung tissues and 16HBE cells using the InSitu Cell Death Detection kit (Roche, Switzerland) according to the manufacturer’s instructions. The nucleus is counterstained with DAPI fluorescent dye (Beyotime BioTech, China) for total cell count. A fluorescence microscope (Olympus, Tokyo, Japan) was used to obtain the images and assess the apoptotic cells. The TUNEL positive cells in each slice were calculated by randomly selecting three different fields. Three fields of view were randomly selected for each cell slide, and 3–6 pieces of each group were taken. ImageJ software is used to count the number of cells in pictures and the percentage of apoptotic cells subjected for statistical analysis.
Data were expressed as mean ± standard error of the mean. GraphPad Prism software (version 5.0; GraphPad, San Diego, CA, USA) was used for 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. P < 0.05 were considered statistically significant. All experiments were repeated at least thrice with consistent outcomes.