2.1. Ethical Statement
This study was approved The Six Affiliated Hospital of Guangzhou Medical University. Thirty four blood samples were obtained from The Six Affiliated Hospital of Guangzhou Medical University. Prior to blood collection, patients were required to provide informed consents.
2.2 Microarray Data and Bioinformatics Analyses
GSE10667 microarray data were retrieved from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nih.gov/geo). Raw data were downloaded as MINiML files. The extracted data were normalized by log2 transformation and adjusted p <0.05. The normalize quantiles function of “preprocessCore” in R (version 4.1.1) was used to normalize the microarray data. Conversion of probes to gene symbols was based on the GPL4133 annotation information of normalized data in the platform. In cases of the same dataset and platform but in different batches, the remove Batch Effect function of “limma” in R was used to remove the batch effects. Data preprocessing outcomes were assessed by boxplot. The PCA plot was drawn to illustrate samples before and after batch effects. To determine the functions of selected mRNAs, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene ontology (GO) enrichment analyses were conducted using DAVID tool (http://david.abcc.ncifcrf.gov). Protein-protein interaction (PPI) network analysis was conducted using the STRING tool (www.string‐db.org).
2.3. Animals
Eight-week-old male C57BL/6 mice were purchased from the Experimental Animal Center of Southern Medical University, housed and cared for in a pathogen-free facility at The Six Affiliated Hospital of Guangzhou Medical University Experimental Animal Center. BLM (coolaber, China) were abdominally injected (40 μg/g) at 0, 2, 4, 6 and 8 days. Recombinant Human CILP protein (CRK PHARMA, China) was intravenously administered (1 μg/g) through the tail, twice a week. Thirty-six mice were randomized into six groups (n = 6): control group, BLM (40 μg/g) group 2 weeks, BLM group 4 weeks, BLM group 6 weeks, BLM + CILP (1 μg/g) 4 weeks, BLM + saline (1 μg/g) 4 weeks. After completion of treatment, mice were euthanized and intact lung tissues resected. Hematoxylin–eosin (HE), Masson, elastic van Gieson (EVG), immunohistochemistry (IHC) analyses, and hydroxyproline (HYP) levels were performed on the obtained samples.
2.4. Isolation of Primary Pulmonary Fibroblasts (PPFs) and their Treatment
PPFs were isolated from 8-week-old male C57BL/6 mice, as previously described. Briefly, lungs were washed, minced and digested using 2.5 mg/mL Dispase II (Solarbio, China) and 2.5 mg/mL Collagenase IV (Rockland, USA) at 37°C for 30 min. After digestion, pulmonary tissues were washed using DMEM medium (Gibco, USA), resuspended in Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and incubated in a humidified 5% CO2 atmosphere at 37 ◦C. Passage 2–5 cells were used for various assays. The ALK5-overexpressing pcDNA3.1-plasmids were synthesized by Guangzhou Laisai Biotech and transfected into cells at a concentration of 2 µg/ml using the Lipo3000TM Transfection Reagent (Invitrogen, USA), as instructed by the manufacturer. After 48 h of transfection, cells were subjected to different treatments (TGF‐β1 (5 ng/ml, SinoBiological, China) or (and) CILP (1 µg/ml, CRK PHARMA, China)) before harvesting.
2.5. Western Blotting Assay
Cells were washed using PBS. Suspended cells (in the RIPA lysis buffer) were lysed on ice. An 8% separation gel and a 5% stacking gel were used for electrophoresis. Through the wet transfer method, separated proteins were transferred to nitrocellulose membranes, which were blocked for 1 h using 5% milk and incubated at 4°C overnight with primary antibodies, including COL I (1:1000, Affinity, USA), a-SMA (1:1000, ABclonal, China), CILP (1:1000, Biossl, China), Smad3 (1:2000, Abcam, UN), p-Smad3 (1:2000, Abcam, UN) and GAPDH (1:1000, ABclonal, China). Enhanced chemiluminescence (Affinity, USA) was used for visualization while Quantity One system (Syngene, UN) was used for quantitative analyses of proteins.
2.6. RT-PCR
The TRIzol (Thermo, USA) reagent was used for total RNA extraction from cells, as instructed by the manufacturer. The PrimeScript RT Master Mix (Takara, Japan) was used to synthesize cDNA from the extracted RNA. mRNAs levels were determined via the SYBR green I incorporation method and real-time PCR system (Syngene, UN). Fold changes in relative gene levels were calculated by the 2−ΔΔCt method. The primers used in this study are shown in supplementary table
2.7. Enzyme Linked Immunosorbent Assay
The right lung tissues were homogenized on ice. The levels of HYP in the right lungs were determined by mouse HYP ELISA kit (Jingmei, China) while blood serum CILP concentrations were determined by the human CILP ELISA kit (Jingmei, China), following the manufacturers’ instructions.
2.8. Histological Analyses
Left lung samples from mice tissues were fixed, paraffin-embedded, sliced into 4 μm thick sections, and stained using H&E staining kits (Sigma, USA), Masson’s trichrome staining kits (Absin, China) or EVG staining kits (Baso, China) as instructed by the manufacturers. Images magnified 6100 (NIH, USA) tool was used for semi-quantitative assessment of fibrotic changes with Ashcroft score, as previously reported[11].
2.9. Immunohistochemistry and immunofluorescence analysis
The tissue sections were treated with citrate buffer at 60 °C for 16 h to remove antigens. Immunohistochemical sections were treated for 15 min with hydrogen peroxide and blocked for 1 h using 1% goat serum. The cells were fixed in paraformaldehyde (4%), incubated with Triton-100 (0.5%), and blocked using bovine serum albumin at room temperature for 2 h. The sections and cells were incubated at 4 °C in the presence of primary antibodies against TGF-β1 (1:100, ABclonal, China), CILP (1:100, Bioss, China), α-SMA (1:100, ABclonal, China), p-Smad3 (1:100, Abcam, UN), COL I (1:100, Affinity, USA), vimentin (1:200, Santa Cruz, USA), and ALK5 (1:200, Santa Cruz, USA). Pulmonary samples or cells were washed and incubated for 1 h with matched secondary antibodies. Hematoxylin (Beyotime, China) and DAB (Beyotime, China) stains were used for immunohistochemical analyses while DAPI stain was used to stain the nuclei for immunofluorescence analysis. Integral optical densities (IOD) for TGF-β1, CILP, α-SMA, TGF-β1, and p-Smad3 immunohistochemistry were analyzed and quantified by Image Pro Plus 6.0 (Media Cybernetics, USA).
2.10. Statistical Analysis
Data are shown as mean ±S.D and were analyzed by the SPSS 25.0 software (SPSS, USA). GraphPad Prism 7.1.0 software (GraphPad, USA) was used to generate graphs. Comparisons of means between and among groups were conducted by the Student’s t-test and one-way ANOVA, respectively. The threshold for significance was p≤ 0.05.