Reagents and instruments
Reagents: Bleomycin sulfate (MCE, Product number:HY-17565); Cyclophosphamide (Manufacturer: Shanghai Yuanye Biotechnology Co., LTD., Item number:S3056); Methylprednisolone (Pfizer, product number: H20130301); Purified Mouse Anti - Rat Granulocytes monoclonal antibody (manufacturer: BD company, the article number 554905); APC Goat Anti-Mouse Ig polyclonal antibody (BD Company, Product number 550826); FITC anti-Rat CD45, PE anti-Rat CD4, APC anti-Rat CD3, PerCP anti-Rat CD8, RBC Lysis Buffer (10X), Cell Staining Buffer(FBS) Cell washes were purchased from BioLegend.
Instruments: micropipettor (Eppendorf, Germany), low-speed horizontal centrifuge (Anhui Zhongke Zhongka, Anhui), flow cytometer Cyto FLEX S (Beckman, USA) Model: BD LSRII, USA. Low speed centrifuge (Model: China Jingli LD5-10B)
Animals And Ethics Statements
Sprague-Dawley rats(male,6–8 weeks of age,160 ± 10g) 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 12h-light/12 h-dark cycles.Rats were divided into four groups(n = 10):(i) blank group, (ii) bleomycin (BLM) group,(iii)BLM-MP group and (iv)BLM-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 intraperitoneal injection of 2.5% sodium pentobarbital (45 mg/kg). They were then fixed on rat plates and sterilized by conventional methods. Bleomycin saline (5 mg/kg, 0.05 mL) was injected into the bifurcation of the trachea. The equal volume of normal saline 0.05 mL was injected into the trachea in the sham operation group. The needle was left in the lung for 6 s and the rats were massaged to distribute the drug evenly in the lung. The model was successfully established on the second day. Drug intervention began 7 days after model establishment. The BLM-MP group was intraperitoneally injected with MP (3 mg/kg) every day, the BLM-MP + CTX group was intraperitoneally injected with MP (3 mg/kg) and CTX (8 mg/kg) every day, and the blank and BLM groups were injected with the same volume of normal saline every day. The animals were sacrificed after 3 weeks of drug administration.
Pathological Staining
The pulmonary tissues of rats were fixed in 4% paraformaldehyde for 24h. After dehydrated, samples were embedded in paraffin, and sectioned to obtain 5µm 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 30min incubation with sections at room temperature. Lastly, sections were developed with 3’3’-diaminobenzidine (DAB, Beyotime, China) for 10min 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 1h, then, with 1% osmic acid for 1.5h. 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).
Ashcroft Scale
According to the scale defined by Ashcroft et al. If there is any difficulty between two odd grades, the field will be given an even score in the middle. In each region, the major degree of fibrosis was recorded as accounting for more than half of the area of the region. The modified scale is defined according to the reference. [22].
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.
Measurement of oxidative stress relating indicators
The homogenate supernatant of the lung tissues was used to detect the MDA content, MPO, GSH-PX and SOD activities by commercial kits (MDA:E-BC-K025-M;MPO:E-BC-K074-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 (#201412, BioLegend, San Diego, CA, USA), CD4 (#201505, BioLegend, San Diego, CA, USA), CD8 (#201712, BioLegend), CD27 (#124207, BioLegend, San Diego, CA, USA), CD28 (#200908, BioLegend, San Diego, CA, USA) 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. CD3 gating was performed according to the side light scattering area (SSC-A) and forward light scattering area (FSC-A). CD27, CD28, and CD3 were detected as the next gating cell population of CD4 and CD8. On the basic of CD3+ CD4+, The proportion of CD27 and CD28 cells was determined.
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.