Eight-week-old male C57BL/6 mice were purchased from Shanghai Laboratory Animal Company, (Shanghai, China). The mice model was established as previously described . Briefly, ten mice in COPD group were exposed to five cigarettes (Nanning Jiatianxia unfiltered cigarettes, 12 mg of tar and 0.9 mg of nicotine) four times per day with 30 min smoke-free intervals in a closed 0.75-m3 room, 5 days per week for up to 12 or 24 weeks. Mice tolerated CS (cigarette smoking) exposure without evidence of toxicity (carboxyhemoglobin levels ∼10% and no weight loss). An optimal smoke:air ratio of 1:6 was obtained. Ten mice in control group were exposed to air. All experiments were repeated three times.
Animal experiments were approved by the Institutional Animal Care and Use Committee of Guilin Medical University and conform to National Institutes of Health guidelines for the use of rodents. This study was reported in accordance with ARRIVE guidelines.
Histology, immunohistochemistry and immunofluorescence staining for lung tissues
Formalin-fixed and paraffin-embedded lung tissues of mice were cut in sections, which were stained with hematoxylin and eosin (HE), Periodic acid–Schiff (PAS) and MASSON to respectively evaluate airway inflammation, goblet cell hyperplasia and mucous secretion, and extracellular matrix in the lung tissues .
For immunohistochemistry, lung-tissue slides were incubated with primary rabbit anti-mouse antibody against α-SMA (dilution 1:200, ab5694; Abcam, Cambridge, UK), C-EBPβ (1:200, ab53138; Abcam, Cambridge, UK), or rat anti-mouse antibody against IL-17 (1:200, ab118869; Abcam, Cambridge, UK) for overnight at 4 °C, and then were incubated for 30 min at 37 °C each with horseradish peroxidase conjugated anti-rabbit or anti-rat IgG antibody and subsequent rinses in PBS three times for 5 min. 3′3-diaminobenzidine-tetrahydrochloride was applied as a chromogen for 1–5 min. Sections were counterstained in haematoxylin for 5–10 min.
For immunofluorescence staining, lung-tissue slides were incubated with primary mouse anti-mouse antibody against E-cadherin (1:100, ab76055; Abcam, Cambridge, UK), or rabbit anti-mouse antibody against Vimentin (1:100, ab92547; Abcam, Cambridge, UK) at 4 °C overnight, and then were incubated with Alexa Fluor 594-conjugated Goat Anti-Mouse IgG(H + L)(SA00006-3, Proteintech, IL, USA) or Alexa Fluor 594-conjugated Goat Anti-Rabbit IgG(H + L)(SA00006-4, Proteintech, IL, USA). Finally, slides were staining with DAPI (4′,6-diamidino-2-phenylindole) at 37 °C for 10 min. Micrographs were obtained using a microscope (BA210T, motic, Xiamen, China).
Health mice were anaesthetized using 2% isoflurane inhalation and killed by cervical dislocation. The bronchus was isolated from lobes of lung, minced to small pieces and digested by 0.05% pronase (Sigma, MA, USA) in DMEM/F12 media (Invitrogen, CA, USA) at 4 °C overnight. Digestion was stopped by adding FBS (Gibco, CA, USA). The bronchial epithelial cells were identified by CK-18 immunofluorescence staining.
Murine bronchial epithelial cells were cultured for 24 h, and then were cocultured with cigarette smoke extract (CSE) or/and IL-17A. CSE was prepared as previously reported . Briefly, filtered cigarettes (Nanning Jiatianxia unfiltered cigarettes, Guangxi, China) were smoked using a peristaltic pump (VWR International) after cutting the filters. Each cigarette was smoked with a 1.5 cm butt remaining. Four cigarettes were bubbled through 40 ml of cell growth medium, and this solution, regarded as 100% strength CSE. In CSE group, bronchial epithelial cells were induced by 20% CSE. In IL-17A group, the cells were induced by 50 ng/ml IL-17A (Biolegend, CA, USA). In CSE + IL-17A group, the cells were cocultured with 20% CSE and 50 ng/ml IL-17A (Biolegend, CA, USA). Cells in all groups were cultured at 37 °C with 5%CO2 for 72 h. The cells without administration of CSE or IL-17A were control group.
Transfection of small interfering RNA (siRNA)
The C-EBPβ siRNA and scramble siRNA were synthesized by Ribobio (Guangzhou, China). The siRNAs were transfected with riboFECTTM CP Reagent according to the manufacturer’s protocol. Bronchial epithelial cells WT mice were transiently transfected with 20 nM scramble or siRNA. After transfection for 24 h at 37 °C in 5% CO2, the cells were prepared as IL-17A group, CSE group, CSE + IL-17A group and controls as described above. The inhibition of C-EBPβ was identified by western blotting.
Quantitative real‑time PCR
Total RNA from lung tissues and cells were respectively extracted using the TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol, and RNA was reverse transcribed into cDNA (Fermentas, Ontario, Canada). A quantitative real-time polymerase chain reaction (PCR) was performed with PIKO REAL 96 instrument (Thermo, MA, USA) and the SYBGREEN PCR Mix Mix (Applied Biosystems, CA, USA) in accordance with the manufacturer’s protocol. Each set of experiments was repeated three times. The 30 μl PCR reactions (with 2 μl cDNA, 0.5 μl (10 μM) forward and 0.5 μl (10 μM) reverse primers, 15 μl SYBR green, and 12 μl PCR H2O) were undergone 10 min at 95 °C, then 40 cycles of 15 s at 95 °C and 60 s at 60 °C. The primers were as follows: mouse E-Cadherin F, 5′-GCAGTTCTGCCAGAGAAACC-3′, and R, 5′-TGGATCCAAGATGGTGATGA-3′; mouse Vimentin F, 5′-GCCAACCTTTTCTTCCCTGA-3′, and R, 5′-TCAAGGTCATCGTGATGCTG-3′; mouse β-actin F, 5′-GATCTGGCACCACACCTTCT-3′, and R, 5′-CTTTTCACGGTTGGCCTTAG-3′. The level of mRNA expression was reported as fold change using the 2−ΔΔCt method.
Western blot analysis for cells
The bronchial epithelial cells were treated with 200 μl RIPA for 10 min on ice, and then were centrifuged at 12,000×g (4 °C) for 15 min. The loaded proteins (50–170 μg) were separated on a 10% SDS-PAGE, followed by transferring onto PVDF membranes. The samples were blocked with TBS-Tween 20 (TBST) containing 5% skim milk for 60 min at room temperature, and at 4 °C overnight, then 30 min at room temperature. The membranes were incubated with rabbit anti-mouse antibody against mouse C-EBPβ (1:1000, ab53138; Abcam, Cambridge, UK), and mouse anti-mouse antibody against β-actin (1:5000, 60008-1-Ig; Proteintech, IL, USA) for 90 min at room temperature, and then they were incubated with horseradish peroxidase conjugated goat anti-rabbit (1:6000, SA00001-2; Proteintech, IL, USA) or anti-mouse antibody (1:5000, SA00001-1; Proteintech, IL, USA) for 90 min at room temperature. At last, blots were developed with the ECL Plus reagents (Thermo pierce, IL, USA).
Immunohistochemistry and immunofluorescence staining for cells
For immunohistochemistry, the slides of cells were incubated with primary rabbit anti-mouse antibody against IL-17R (1:50, ab180904; Abcam, Cambridge, UK) at 4 °C overnight, and then were incubated with horseradish peroxidase conjugated goat anti-rabbit IgG antibody (PV-9000, Zisbio, Beijing, China) at 37 °C for 30 min. After rinsing with PBS for three times, 3′3-diaminobenzidine-tetrahydrochloride was applied on the slides as a chromogen for 1–5 min. Slides were counterstained in haematoxylin for 5–10 min.
For immunofluorescence, the slides of cells were incubated with primary mouse anti-mouse antibody against Cytokeratin18 (1:50, ab668; Abcam, Cambridge, UK), mouse anti-mouse antibody against E-cadherin (1:50, ab76055; Abcam, Cambridge, UK), or rabbit anti-mouse antibody against Vimentin (1:50, ab92547; Abcam, Cambridge, UK) at 4 °C overnight, and then were incubated with Alexa Fluor 594-conjugated Goat Anti-Mouse IgG(H + L)(SA00006-3, Proteintech, IL, USA) or Alexa Fluor 594-conjugated Goat Anti-Rabbit IgG(H + L)(SA00006-4, Proteintech, IL, USA). Finally, slides were stained with DAPI (4′,6-diamidino-2-phenylindole) at 37 °C for 10 min.
Group data were expressed as the mean ± standard deviation (SD). Significant differences were evaluated using t-test. P values < 0.05 were considered to be statistically significant. Statistical analyses were performed using SPSS 21.0 (IBM SPSS Inc., Chicago, IL, USA).