Animal Experiments
C57BL/6 mice were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine. Animal experiments were performed following the Declaration of Helsinki Convention on the Use and Care of Animals and were approved by the Animal Research Ethics Committee of Guangzhou Medical University.
Collection and Extraction of PM2.5
Traffic ambient PM2.5 was collected by aerodynamic impactors equipped with a glass fiber filter, quartz filter, or teflon membrane, DMSO was used to extract soluble parts of PM2.5. Sufficient specific samples for biological experiments were collected by high volume impactors equipped with glass fiber filter from arterial traffic road. Gravimetric analysis was used to analyze specific physicochemical characterizations of PM2.5. The procedure of PM2.5 samples and analysis methods were obtained as our previously described [10].
PM2.5 exposure in mice
Female C57BL mice(6-8w) were randomly divided into 4 groups of 12 mice each. PBS (20 μl), PM2.5 (100 μg/20 μl), BOX5 (0.5 μg/ml 10 μl), and PM2.5+BOX5 (100 μg/20 μl+ 0.5 μg/ml 10 μl) were injected via tracheal drip, respectively, twice a week for 4 weeks of continuous exposure. Collection of lung tissue for follow-up experiments.
Lung function tests
Four weeks later, mice were anaesthetized intraperitoneally with 1.25 % avertin (2,2,2-tribromoethanol) 24 hours after the last exposure to PM2.5. Tracheotomized mice were placed in a whole-body manometer (Buxco-Force Pulmonary Maneuvers) to determine the peak expiratory flow rate (PEF) and peak inspiratory flow rate (PIF) by Boyle's law maneuvers.
Histology and Immunohistochemistry
Mice lung tissues were collected and analyzed for small airways by histochemistry and immunohistochemistry according to our previous descriptions[17, 18]. The histology was performed on 4.0 μm thick paraffin sections of lung tissues. Sections were incubated with Anti-α-SMA antibody. (ab5694, Abcam). Immunochemistry of α-SMA positive stained area was evaluated with the confocal microscope at 200× magnification.
Analysis method of airway remodeling
The thickness of the small airway wall was the total area of the small airway wall (WAt) after normalization of the perimeter of basement membrane(Pbm), i.e., WAt/Pbm (μm2/μm), which was used as an index of small airway remodeling. The requirement of airway was selected as follows: 1. basement membrane perimeter < 2000 μm; 2. minimum internal diameter of airway/maximum internal diameter of airway > 0.5; Pbm, area of basement membrane(Abm) and airway wall outer membrane area (Ao) were measured using Image pro-plus 6.0 (IPP6.0) software; The percentage of smooth muscle layer area (smooth muscle specific marker protein α-SMA positive area) to airway wall area, i.e. Area/(Ao-Abm)% (WA%), was used as an indicator of smooth muscle layer in airway remodeling.
Analysis method of emphysema
The mean linear intercept (MLI) was used to evaluate the size of the alveolar cavity, which was used as an index of emphysema. At the same field of view, "十" intersecting lines were drawn, and the length of each line (L) and the number of alveoli passing through each line (NS) were calculated, and MLI was expressed as MLI=L/NS.
Cell culture and treatment
The human bronchial smooth muscle cell line (HBSMC; American Type Culture Collection, Manassas, VA, USA) was cultured in SMCM (sciencell, USA). Cells were cultured at 37°C with 5% CO2 with humidification and then exposed (or not) to PM2.5 (3 μg/ml). HBSMC were pretreated with the Wnt5a antagonist BOX5 (Merck Millipore, Burlington, MA, USA) for 1 h prior to the addition of 3 μg/ml PM2.5.
Cell-viability assay
The viability of HBSMC was detected using the WST8 assay kit (CCK8; Promoter Biotechnology, Wuhan, China) according to the manufacturer's instructions. Cells were exposed to BOX5 (100, 200,300 μM) in SMCM culture medium (100 μl). The optical density (OD) at 450 nm was detected with a microplate reader (Multiskan MK3; Thermo Fisher Scientific, Waltham, MA, USA).
Immunofluorescence
HBSMCs were cultured for 48 h to reach 60% density. Then immunofluorescence was performed following standard procedures. Cells were incubated with Anti-β-catenin antibody (ab32572, Abcam, 1:500) for 12 h at 4°C, followed by goat anti-Rabbit IgG-antibody (ab150077, Abcam, 1:400) for 1 h at room temperature. Finally, cell nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China) for 5 min. Images were examined using an inverted fluorescence microscope (Olympus-FL 500; Olympus Corporation, Tokyo, Japan).
Isolation of mRNA and real-time PCR analysis
Total mRNA was extracted using the Universal RNA Extraction Kit (B0004D, EZB, Shanghai, China) according to the manufacturer's instructions. mRNA was quantified using a NanoDrop spectrophotometer (NanoDrop Tech, Rockland, DE, USA). Gene-specific primers (listed in Table 1) were provided by Sangon Biotech (Shanghai, China). Quantitative real-time PCR was performed using a SYBR Premix Ex Taq II (Novozymes) and a Bio-rad instrument II (Roche Diagnostics, Basel, Switzerland). GAPDH served as the housekeeping control.
Table 1
Primer
|
Primer sequence
|
Human Wnt5a
|
F:5’-ATTCTTGGTGGTCGCTAGGTA-3’
R: 5’-CGCCTTCTCCGATGTACTGC-3’
|
Human β-catenin
|
F:5’-GGAATCCGAGCTGGACCTAAA-3’
R: 5’-CCTGAAGCAAATCGACCACAG-3’
|
Human TGF-β1
|
F:5’-GGGATTGGCTGTATGAGCACC-3’
R: 5’-GGCGGGAAATTGTGAACTGA-3’
|
Human c-myc
|
F:5’-CACCTTGTAGCACGTCCTG -3’
R: 5’-GACTCCCCAAGATGTGGTGG-3’
|
Human CyclinD1
|
F:5’-CAATGACCCCGCACGATTTC -3’
R: 5’-CATGGAGGGCGGATTGGAA-3’
|
Human GAPDH
|
F:5’- TGTGGGCATCAATGGATTTGG-3’
R: 5’-ACACCATGTATTCCGGGTCAAT-3’
|
Protein extraction and Western blot analysis
Protein extraction and Western blotting was conducted following the manufacturer’s
instructions. The membranes were blocked with 5% BSA (G5001, Servicebio) and then incubated with antibodies (Anti-PCNA antibody, ab18197, Abcam; Anti-α-SMA antibody, ab5694; Anti-Wnt5a antibody, ab179824; Anti-β-catenin antibody, ab32572; Anti-PDGFRβ antibody, ab69506; Anti-TenascinC antibody, ab108930; Anti-α-Tubulin, ab7291; Anti-β-Actin, ab8226, Abcam;) at 4°C for 12 h. Subsequently, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 hours at room temperature. The proteins of interest were detected by enhanced chemiluminescence reagents (FDbio science), and the band intensities were quantified by using the ImageJ software (National Institute of Health, Bethesda, MD, USA). The expression of the target proteins was normalized against the loading control, β-actin or α-tubulin.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 7.04 for windows (GraphPad Software, San Diego, USA) and are presented as median and IQR. Data was analyzed with a one-way ANOVA with Bonferroni post-hoc test or Kruskal–Wallis with Dunn’s test for multiple comparison depending on respectively parametric or non-parametric datasets. p<0.05 was considered statistically significant.