45BALB/c female mice (free of specific pathogens, 6–8 weeks, weighing 20-22g) were supplied from animal feeding center of Yanbian University. Breeding condition was 50–60% relative humidity, room temperature at 22 ± 2°C, 12h alternating day and night. All animal experimental procedures were applied in accordance with the Regulations for the Administration of Laboratory Animals and approved by the Ethics Committee of the College of Medicine of Yanbian University (approval number SYXK (JI) 2020-0009). All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
Animal model and grouping
Mice were randomly divided into 5 groups (n = 9), control group, cockroach extract (CRE) group, ginsenoside Rb1 treated (low/high dose) group, and dexamethasone group. The mice in the CRE group were each treated with CRE (50 µg, Greerlabs, XP46D3A4, German) dissolved in 50 µL of saline on day 1 to day 5 via intranasal (i.n.) administration for sensitization. 50 µL of saline was applied for control group mice. On day 11 to day 15, mice were continuously stimulated with equivalent volume and concentration of CRE (i.n.) daily under light anesthesia[19]. Mice in the Rb1-treated group were gavaged with Rb1 (10 and 20 mg/kg, B21050, yuanye Bio-Technology Co. Shanghai, China) for 5 d, 1 time/d[14]. The control group used 50 µL saline. Mice were sacrificed by cervical dislocation under deep anesthesia using halothane 2 hours after the last challenge for further analysis.
Sample collection
BALF was obtained from 3 randomly selected mice in each group, then were stained with Diff-quick (G1541, Solarbio, Beijing, China) to calculate the proportion of eosinophils. Other 3 mice were extracted for serum analysis form peripheral blood, mediastinal lymph nodes (mLNs) and right lung for molecular biology analysis. The left lungs were applied by histochemical staining. The rest of lungs were frozen for further analysis.
ELISA
The cytokine levels from BALF supernatant were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA), including IL-4, IL-5, IL-13 and IFN-γ (M4000B, M5000, M1300CB, MIF00). All with a sensitivity of 2.0pg/mL. Serum CRE-specific and total IgE (SEKR-0019, Solarbio) were also measured[20]. MDA, CAT, SOD (A003-1-2, A007-1-1, A001-3-2; Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China) ELISA assay was performed.
Histological analysis
Paraffin embedded lung sections were stained with Hematoxylin and Eosin Kit (G1120, Solarbio) to observe airway inflammation. Modified Masson's Trichrome Stainning (G1346, Solarbio) was performed to observe collagen deposition.
MTT assay
The BEAS-2B cells viability after treatment was applied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) solution as previously described[21].
Cell culture and treatment
The BEAS-2B cells were purchased from the Cell Resource Centre, Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China). The cell culture medium was DMEM (vivacell) dissolved with 10% fetal bovine serum (vivacell, Shanghai, China), streptomycin (100 g/mL) and penicillin (100 U/mL), incubated at 37°C and 5% CO2. For stimulation, 1 × 105 of cells were administered with 60µM of Rb1 for 1h[22], or treated with MitoTempo (10µM, HY-112879, MCE, USA) for 30 min[23], or with SIRT1 agonist SRT1720 (HY-15145, 1µM, MCE) for 6 h[24], and then exposed to 50µg/ml of CRE for 24h[19].
ROS, mitochondrial morphology, mitochondrial membrane potential assay (MMP), TUNEL
Total cellular ROS was detected by incubation with DCFH-DA (10 µM, S0033S, Beyotime) fluorescent probe at 37°C, 10 min. Tissue ROS was detected by Dihydroethidium (DHE) (10 µM,S0063,Beyotime) at 37°C, 5 min. For mitochondrial morphology, intact tubular networks of mitochondria were characterized as not injury, on the contrary, ruptured and spherical morphology of mitochondria defined as fission mitochondria. Mitochondrial length and the DRP1 density were all analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). Cellular mitochondrial ROS (mtROS) was detected by MitoSOX (dye, 5µM, M36008, Thermo Fisher Scientific, Massachusetts, USA) for 10min. For MMP detection, cells were stained with JC-1 (5µM, C2006, Beyotime) at 37°C for 15min and calculate the red/green fluorescence intensity ratio. Apoptosis was detected using the Dead End fluorescent TUNEL kit (C1089, Beyotime). All images were photographed using the Cytation5 Cell Imaging Microplate Detection System (BioTek, Inc., Winooski, VT, USA).
Western blotting
After extraction, proteins (20 µg) was applied by 12% SDS-PAGE. The gel was incubated with antibodies: SIRT1 (110304, Abcam, USA), Cleaved Caspase-3 (32042, Abcam), PGC-1α (77210, Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 5174, CST, USA), Bax (182734, Abcam), DRP1 (ab184247, Abcam), Bcl-2 (182858, Abcam), MFN1 (221661, Abcam) and p-DRP1 (Ser616) (3455, CST, USA), PI3K(4292, CST), p-PI3K(17366, CST), AKT (8805, Abcam) and p-AKT(38449, Abcam) for 4℃ overnight. Then incubated with antibodies HRP-goat anti-rabbit antibody (5151, CST, USA) or HRP-anti-mouse antibody (5257, CST). Grey-scale values were measured using Quantity One software (BioRad, Hercules, CA, USA).
Flow cytometry
BAL cells and single cells suspension of mLNs were incubated by surface marker FITC-CD4 antibody (11-0041-82, Invitrogen, USA). Cells were then treated with Intracellular Fixation and Permeabilisation Kit (88-8824-00; Invitrogen), followed by staining by APC-IL-4 antibody (554436, BD, USA) and PE-CY7-IFN-γ antibody (25-7311-82, Invitrogen, USA). Samples were finally collected using a CytoFLEX flow cytometer (Beckman Coulter, Inc., CA, USA) and calculated for the ratios of IL-4+ and IFN-γ+ in the CD4+ cells using Cytoexpert 2.4 software.
Immunofluorescence assay
For mitochondrial imaging, treated cells were incubated with the mitochondrial marker MitoTracker Red (M7512, Thermo) at 37°C, 30 min. For co-staining of MFN1 and mitochondria, lung sections firstly incubated with MitoTracker Red, washed and incubated at 4°C (overnight) with rabbit anti-MFN1 antibody (57602, Abcam). For the DRP1 translocation assay, cells were stained with MitoTracker Red and then incubated with rabbit anti-DRP1 antibody (8570S, CST). At last, tissue sections were treated by DAPI (P0131, Beyotime). The slides were photographed by Cytation5. Fluorescence density was analyzed by Image J.
Statistical analysis
Data are applied as the mean ± standard deviation of three independent experiments. Significance of differences between the two groups was determined by Student t-test with SPSS 19.0 software (IBM Co., Armonk, NY, USA). Multiple comparisons were performed using ANOVA or Wilcoxon rank sum test. We considered statistically significant when the p-value was < 0.05.