Animals and ethics statements
Sprague-Dawley rats (male, 6-8 weeks of age, 160 ± 10 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Rats were performed a week of adaptation before starting the study. Rats were allowed to eat normal pellet feed and tap water freely throughout the study period, and maintained under standard temperature (22 ± 2 °C) and relative humidity (55 ± 5%) with 12 h-light/12 h-dark cycles. Rats were divided into four groups (n = 10): (i) sham group, (ii) PF group, (iii) PF + MP group and (iv) PF + MP + CTX group.All animal experiments were reviewed and approved by the Animal Ethics Committee of the Beijing Medconner Biotechnology Co., LTD and conform to National Institutes of Health guidelines for the use of rodents. This study was reported in accordance with ARRIVE guidelines.
Animal modeling and treatment
Rats were anesthetized by administrating (i.p.) 2.5% Pentobarbital Sodium (45 mg/kg). Then, they were fixed on the rat plate and disinfected with conventional methods. Rats were injected with a special needle for tracheal spray from the left corner of the mouth of the rats and placed near the trachea, bleomycin in normal saline solution (5 mg/kg, 0.05 mL) was injected into the bifurcation, and equal volume normal saline (0.05 mL) was injected into the trachea of the sham group. The needle stayed in the lung for 6 s and massaged rats to make the drug evenly distributed in the lungs. MP (3 mg/kg) was given (i.p.) every day in PF + MP group, and MP (3 mg/kg) and CTX (8 mg/kg) were given (i.p.) daily in PF + MP + CTX group for 21 days. The PF group and sham group were injected with the same volume of normal saline every day.
Pathological staining
The pulmonary tissues of rats were fixed in 4% paraformaldehyde for 24 h. After dehydrated, samples were embedded in paraffin, and sectioned to obtain 5 μm thickness sections. Then, hematoxylin and eosin (H&E) and Masson’s trichrome staining were performed according to the manufacturer’s instructions (Nanjing Jiancheng, China). For immunohistochemistry, sections were incubated with primary antibody against α-SMA (1:3000, 14395-1-AP, Proteintech, China) and collagen I (1:1000, 14695-1-AP, Proteintech) at 4 °C overnight. Negative control was obtained by ignoring the incubation with primary antibody. After that, Goat anti-rabbit HRP labeled secondary antibody (1:1000, ab6721, Abcam, USA) was used for 30 min incubation with sections at room temperature. Lastly, sections were developed with 3’3’-diaminobenzidine (DAB, Beyotime, China) for 10 min and images were acquired under a light microscope (Leica, Germany).
Transmission electron microscope
The lung tissues were fixed with the mixture of 2.5% glutaraldehyde and 2% polyformaldehyde at 4 °C for 1 h, then, with 1% osmic acid for 1.5 h. Then, tissues were dehydrated with gradient alcohol, embedded and sliced. Subsequently, sections were stained and observed by a H-7500 transmission electron microscope (Hitachi, Japan).
Western blot
Total proteins were extracted from lung tissues using radio immunoprecipitation assay (RIPA) buffer with proteinase inhibitor (Solarbio). 40 μg proteins of each sample were separated by 10% SDS-PAGE and proteins were transferred to PVDF membranes (Millipore). The membranes were sealed in 5% skim milk for 1 h at room temperature and incubated with primary antibodies, GAPDH (1:50000, 60004-1-Ig, Proteintech), α-SMA (1:4000, 14395-1-AP, Proteintech) and collagen I (1:2000, 14695-1-AP, Proteintech) at 4 ℃ overnight. Next, membranes were washed with tris buffer containing 0.05% twin-20 (TBST) and incubated with secondary antibody (1:10000, ab6721, Abcam) for 1 h at room temperature. Immunoblots were visualized by an ECL kit (Abcam) followed by quantitative analysis via ImageJ software.
Enzyme linked immunosorbent assay (ELISA)
TNF-α (EK382/3-02, Multi Sciences, China), IL-1β (EK301B/3-02, Multi Sciences) and IL-6 (EK306/3-01, Multi Sciences) levels in serum and bronchoalveolar lavage fluid (BALF) were detected by ELISA, following the manufacturer’s guidelines.
Measurement of oxidative stress relating indicators
The homogenate supernatant of the lung tissues was used to detect the malondialdehyde (MDA) content, glutathione peroxidase (GSH-PX) activities and superoxide dismutase (SOD) by commercial kits (MDA: E-BC-K025-M; SOD: E-BC-K020-M; GSH-PX: E-BC-K096-M, Elabscience Biotechnology Co., Ltd, China) according to the instructions.
Flow cytometry
Isolated single cells from rat BALF were stained with CD3 (#201414, BioLegend, San Diego, CA, USA), CD4 (#201509, BioLegend), CD8 (#200610, BioLegend), CD27 (#124212, BioLegend), CD28 (#554993, BD Biosciences, San Jose, CA, USA), CD25 (#202114, BioLegend), FOXP3 (ab215206, Abcam) antibodies for 30 min at 4 °C. Then, Flow cytometry was performed by using a Beckman CytoFLEX device and FlowJo software was used for analysis.
Statistical analysis
The measurement data were expressed as mean ± standard deviation (SD), one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used for multiple group comparison by using Graphpad Prism. Differences at p <0.05 were considered statistically significant.