Chemicals
Bixin (> 97% purity) purchased from Med Chem Express (MCE, USA) was dissolved in DMSO or coin oil according to the experiments purpose. Aluminum hydroxide adjuvant was purchased from Imject® Alum, Pierce (USA). Ovalbumin (OVA) and Complete Freund’s Adjuvant (CFA) was purchased from Sigma-Aldrich (China). TGF-β1 was obtained from RD System (Minneapolis, MN).
Cell culture
The mouse airway epithelial cell line MLE-12 cells were procured from the China Cell Collection Center (Beijing, China) and maintained in Dulbecco’s Modified Eagle medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (Gibco) in a humidified atmosphere of 5% CO2 at 37°C. The culture medium was replenished every three days.
Animals
Six-week-old Balb/c female mice were supplied by the Liaoning Changsheng Biotechnology Co., Ltd (Liaoning, China). All animals were raised in enclosures of uniform temperatures (24°C ± 2°C) and humidity (45%). All animal experimental procedures were approved from the Ethics Committee of Medical Experiment Animals in the College of Basic Medicine of Jilin University.
Establishment of an acute asthma model
An acute murine asthma model was established as described before [9]. Briefly, 1mg aluminum hydroxide and 20 µg OVA were dissolved in 200 µl sterile phosphate-buffered saline (PBS). The solution was injected intraperitoneally (i.p.) to mice for sensitization on day 0, 7, and 14. The same volume of PBS only was administered in the same manner into control mice. From day 21 to 23, the mice were subjected to nebulized OVA (1% OVA in PBS) for 30min daily. As controls, mice were injected and nebulized with PBS. For the treatment, Bixin (50mg/kg or 100mg/kg, dissolved in corn oil) was given via i/p. injection daily from day 18 to day 23. All animals were sacrificed on day 25 after anesthetization. The experimental procedure is outlined in Fig. 1A.
Establishment of a chronic asthma model
Experimental and control group mice were sensitized following the same protocol used to establish acute asthma models. From day 17 to 58, mice were treated thrice a week with 30min of nebulized OVA (1% OVA in PBS) [29]. Mice of the control group were given nebulized PBS alone. Bixin was given from day 45 till day 58 and mice were sacrificed on day 59. The experimental procedure is outlined in Fig. 4A.
Establishment of a GCs-resistant asthma model
To establish a GCs-resistant asthma model [30], mice were injected subcutaneously on day 0 with CFA emulsified OVA. From days 21–23, mice were exposed to aerosolized OVA (1% OVA in PBS) for 30min daily. Mice were treated with Bixin (100mg/kg), dexamethasone (5 mg/kg i.p. injection) or both from day 18 till day 23 and mice were sacrificed on day 25. The experimental procedure is outlined in Fig. 3A.
Assessment of airway hyperresponsiveness (AHR)
Non-invasive whole-body plethysmography (Model PLY 3211; Buxco, Sharon, CT, USA) was used to measure AHR at 24h after the last challenge. In brief, conscious and spontaneously breathing mouse was placed in a chamber for measurement of pressure/time waves by the connected sensor and related computer data acquisition system. The degree of AHR was presented as Enhanced Pause (Penh) values. The mice were treated with different concentrations of nebulized methacholine (2.5, 5, 10, 25 and 50mg/ml; Sigma-Aldrich), and Penh values were recorded with each dose.
Bronchoalveolar lavage fluid (BALF)
After euthanasia, mice trachea was exposed via blunt dissection. The trachea was incised and a small-caliber tube was placed into the airway. Sterile ice-cold PBS (1ml) was then used to lavage the lungs twice. BALF cells were pelleted by centrifugation and the supernatant was stored in -80°C. The cell pellets were resuspended with cold PBS, followed by spun onto glass slides for Diff Quick staining (Soledad Bao, Beijing, China).
Cytokine quantification
Protein levels of interleukin-5 (IL-5), IL-13, IL-1β, IL-6, IL-17A, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) were measured by enzyme linked immunosorbent assay (ELISA) using commercial kits (Elabscience, Wuhan, Hubei, China) according to the instruction manual.
Collagen content measurement
Lung tissue collagen content from the various experimental groups were quantified via a Sircol assay kit (Biocolor, Carrickfergus) in accordance with manufacturer protocols.
Hematoxylin and eosin (H&E), PAS and MASSON staining
Briefly, the left lungs of sacrificed mice were harvested and fixed with 4% formaldehyde, followed by embedded in paraffin. The lung tissue was cut into 4-µm sections, followed by H&E, PAS and MASSON staining in compliance to standard manufacturer protocols (Soledad Bao, Beijing, China).
Immunohistochemistry
The lung paraffin sections warmed overnight in a 60°C oven. Sections were dewaxed, hydrated and boiled in 1 × sodium citrate (PH = 6) and cooled to room temperature for antigen retrieval. After blocking endogenous peroxidases and nonspecific staining, the slides were incubated sequentially with p65 (Proteintech), E-cadherin, N-cadherin and 𝛂-SMA (Abclonal) overnight in a wet box at 4°C. The following morning, a secondary antibody (Abclonal, China) labelled with biotin was used to incubate the slides. Lastly, 3,3′-diaminobenzidine (DAB) chromogen solution was used to stain the section before they were subjected to conventional dehydration, xylene clearing and fixing.
Antibodies and Immunoblotting
Total proteins of the cells or tissues under different conditions were lysed using a 1×RIPA Kit (Beyotime, Shanghai, China). A bicinchoninic acid assay (BCA) kit (Thermo Scientific, Rockford, USA) was utilized to ensure equal protein loading. 40µg of protein was electrophoresed using a 10% SDS-PAGE gel before being blotted onto nitrocellulose membranes. Membranes were blocked for an hour with 5% milk powder and incubated overnight with primary antibodies at 4°C. Primary antibodies against p65, p-IκBα, IκBα (Proteintech), E-Cadherin, N-Cadherin, α-SMA, p-PI3K, PI3K, p-Akt, AKT, p-mTOR, mTOR p-SMAD2, p-SMAD3, SMAD2, SMAD3 and GAPDH (Abclonal) were used. The next day, membranes were rinsed thrice with PBST and incubated at room temperature with secondary antibodies conjugated HRP at room temperature. Samples were then exposed to an ECL reagent (Beyotime, Shanghai, China) after rinsing them thrice with PBST.
Statistical analysis
The GraphPad Prism 6 (La Jolla, CA) was used to carry out data analysis. The one-way or two-way analysis of variance (ANOVA) followed by Turkey’s post hoc test or Dunnett-t post hoc test was used to analyze all results. Statistical significance was designated to results with p-values of less than 0.05.