Reagents and antibodies
NIF was purchased from Xiyashiji Chemical Co., Ltd. (Chengdu, Sichuan, China). For the in vitro study, a 20 mM/50 mM NIF solution was prepared in dimethyl sulfoxide (DMSO) (Sigma, St Louis, Mo), stored at − 20 ℃ and diluted in culture medium to the required concentration. When the DMSO concentration was < 1‰, the culture medium was used as the vehicle control; when the DMSO concentration was ≥ 1‰, the same concentration of DMSO was used as the vehicle control. For in vivo studies, NIF was prepared at a 5:35:60 ratio of DMSO: polyethylene glycol 400 (PEG 400) (Sigma, St. Louis, MO, USA): normal saline and administered at a dose of 0.1 mL/10 g body weight. BLM sulphate was purchased from Chengdu Synguider Technology Co., Ltd. (Chengdu, China). Collagenase Type IV was purchased from Gibco (#17104-019; Grand Island, NY, USA). TGF-β1 was purchased from Novoprotein (#CA59; Shanghai, China). Primary antibodies against β-actin (ab8226; Abcam, Cambridge, MA, USA), GAPDH (TA-08; ZSGB-BIO, Beijing, China), α-SMA (ab5694), Collagen-I (ab88147), E-Cadherin (ab76055) and Vimentin (ab20346) were purchased from Abcam (Cambridge, MA, USA), and Smad2/3/phospho-Smad2/3 (#8685; #8828) and Stat3/phospho-Stat3TY705 (#9139; #9145) were purchased from Cell Signaling Technology Company (MA, USA). PE-CD11b (#12-0112-82), PE-CD4 (#12-0041-82), APC-CD69 (#17-0691-82), and FITC-CD8 (#11-0081-82) were purchased from BD Biosciences (San Diego, CA, USA). PE-F4/80 (#123110), FITC-CD11b (#101206), and FITC-Gr-1 (#108406) were purchased from Biolegend (San Diego, CA, USA).
A549 (human alveolar basal epithelial cells) and NIH/3T3 (mouse embryonic fibroblasts) were purchased from ATCC (Rockville, MD, USA). HPFs (human pulmonary fibroblasts) were purchased from Science Cell (San Diego, CA, USA). The three cell types were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% or 20% heat-inactivated foetal bovine serum (FBS; HyClone, Logan, UT, USA) and 1% penicillin/streptomycin (MP Biomedical LLC) in 5% CO2 at 37 °C.
Molecular docking studies
The three-dimensional X-ray crystal structure of Stat3 (PDB ID: 6NJS) was downloaded from the Protein Data Bank . Both the compound and protein were processed using the CHAR Mm force field . Molecular docking was performed using the CDOCKER module in Accelrys Discovery Studio (version 3.5; Accelrys, San Diego, CA, USA). The molecular docking parameters were determined according to the standard values set by the software. After the docking study was completed, the platform was used to collect the docking score and analyse the docking modes.
The thiazolyl blue tetrazolium bromide (MTT) (Sigma, St Louis, MO) assay was used to evaluate the viability of NIF-treated cells. Cells were cultured in 96-well plates at a density of 1000–8000 cells/well and administered NIF (0–20 μM) 24 h later. Next, after coincubation for 24, 48, and 72 h, 20 μL of 5 mg/mL MTT was added to each well, and cells were further incubated at 37 °C for 2–4 h. A Spectra MAX M5 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) was used to determine the absorbance at 570 nm.
Cells were cultured at a concentration of 10,000–20,000 cells/well in 24-well plates containing glass slides (14 mm × 14 mm). After 48 h of culture, the medium was discarded and replaced with serum-free DMEM for 6 h. Then, the medium was replaced with complete medium, and 5 ng/mL TGF-β1 was added. After 1 h, NIF (20 μM) was added and incubated for another 24 h. Next, the slides were fixed in 4% paraformaldehyde for 15 min at room temperature followed by washing 3 times with PBS and permeabilizing with 0.5% Triton X-100 (Sigma, St Louis, MO) for 20 min. After washing 3 times with PBS, cells were blocked in 5% BSA (BioFroxx) solution at room temperature for 30 min followed by incubation with primary antibodies (anti‐E‐cadherin (1:200; Abcam, Cambridge, MA, USA) and anti-α-SMA (1:200; Abcam, Cambridge, MA, USA)) overnight at 4℃. The slides were subsequently washed 3 times with PBS (5 min each time) and incubated with the secondary antibody (Cy3‐labelled goat anti‐mouse immunoglobulin G (IgG; Beyotime, Shanghai, China) at a 1:200 dilution (red) or FITC-488‐labelled goat anti‐rabbit immunoglobulin G (IgG; Beyotime, Shanghai, China) at a 1:200 dilution (green)) for 1 h at room temperature. The slides were then washed 3 times with PBS and stained with DAPI (Biosharp, Hefei, China) at room temperature for 10 min. After washing 3 times with PBS, an anti-fluorescence quencher (IgG; Beyotime, Shanghai, China) was added. Immunofluorescence was analysed under a fluorescence microscope (Zeiss LS880, Germany).
Wound migration assay
A549 cells were inoculated into 6-well plates at a density of 105 cells/well. When the cells had reached approximately 80% confluence, the medium was discarded and replaced with serum-free medium. After incubation for 6 h at 37 ℃, the cells were scratched using 200 μL pipette tips. The cells were washed with PBS to remove cellular debris and cultured in complete medium (less than 3%) containing TGF-β1 (5 ng/mL). One hour later, NIF (20 μM) was added, and images of the scratches were acquired 0 and 24 h later using a microscope (Olympus, ix73, Japan). The wound closure rate was calculated as the ratio of the cell migration area and the original wound area.
Cell/tissue lysates and western blot
Protein detection were performed in accordance with previous reports . Cells were inoculated into Petri dishes (10 cm) and then treated with the same immunofluorescence procedure as those collected after 24 h of NIF administration. Collected tissue was frozen in liquid nitrogen and ground into a powder in a mortar. Lung tissue and cultured cells were homogenized in RIPA buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (Selleckchem) and quantified using the Bradford method. Then, the proteins were separated on 10% SDS–PAGE gels (Chengdu Baihe Technology Co., Ltd.) and transferred onto 0.45 μM PVDF membranes (Merck Millipore, Billerica, MA, USA). After blocking in 5% (M/V) non-fat milk for 1 h, the membranes were incubated with antibodies overnight at 4 °C. Then, the membranes were incubated with goat anti‐rabbit/mouse IgG (ZSGB-BIO Co., Beijing, China) at a 1:3000 dilution for 60 min at 37 °C. Reactive bands were identified using an enhanced chemiluminescence kit (Merck Millipore, Billerica, MA, USA). Then, the images were analysed using ImageJ software (National Institute of Health, Bethesda, MD, USA).
Thermal drift assay
These methods were performed according to those reported in the literature  with some modifications. Cells were cultured in the same manner described above for protein extraction. After treatment with NIF (200 μM) for 2 h, the cells were collected, PBS containing protease inhibitor was added, and the cells were adjusted to the same concentration after cell counting. The cell suspension was divided into 8 equal parts and heated at 42, 44, 46, 48, 50, 52, 54 and 56 °C for 3 min, frozen in liquid nitrogen (1 min) and re-dissolved at room temperature three times. After centrifuging the samples at 13,300 r/15 min, the final protein was obtained by removing the supernatant. Then, protein images were obtained and analysed according to the method described above.
Isolation and culture of lung fibroblasts
Wistar rats (male; 6–8 weeks; 180–220 g) were purchased from Vital River (Beijing, China). Rats were anaesthetized using 10% chloral hydrate and then intratracheally administered BLM (5 mg/kg). After 14 days, the rats were sacrificed, and complete lung tissue was obtained by dissection. Then, the lungs of the rats were washed with Hanks solution, minced and digested in trypsin. After incubation at 37 °C for 40 min, the cell suspension was filtered through a 70 μM screen mesh. The filtrate was centrifuged at 1500 rpm/5 min and the precipitates were collected, mainly lung fibroblasts and epithelial cells. The precipitate was resuspended in the medium and centrifuged at 800 rpm for 5 min. Take the supernatant (mainly including lung fibroblasts and a few epithelial cells), discard the precipitate (mainly including epithelial cells), centrifuge the obtained supernatant at 1500 r/5 min, and then collect the sediment containing lung fibroblasts [22, 23]. The extracted precipitate containing primary lung fibroblasts were cultured in DMEM/F-12 (GIBCO, NY, USA) medium containing 10% FBS and 1% penicillin and streptomycin and streptomycin in 5% CO2 at 37℃. After culturing in the incubator for 40 min, discard the non-adherent cells (Lung fibroblasts adhere faster and can be separated from other cells) . The adherent cells are pure lung fibroblasts and cultured in fresh culture medium. This method was also used in the subsequent passage to further purify the extracted lung fibroblasts. Lung fibroblasts were identified by morphology and immunofluorescence (Additional file 1: Fig. S1a, b) . The cells were used between passages 3 and 8.
Animal studies of fibrosis
C57BL/6 mice (6–8 weeks) were purchased from Huafukang (Beijing, China). C57BL/6 mice were anaesthetized using 10% chloral hydrate and then intratracheally administered BLM, and the sham-operated group was administered with the same volume of physiological saline.
In the preventive model, mice were intratracheally administered BLM (~ 2.5 mg/kg) while the sham-operated group was administered with the same volume of physiological saline. All bronchial drip only once at day 0. On the first day of BLM bronchial drip, all BLM mice were randomly divided into 3 groups: n = 14 (Vehicle); n = 10 (NIF 25 mg/kg); n = 14 (NIF 50 mg/kg). The sham operation group (n = 10) and the vehicle group were intraperitoneally injected with dissolvent (5:35:60 ratio of DMSO: polyethylene glycol 400: normal saline) every day for 27 days. At the same time, the NIF groups received intraperitoneal injection of low-dose NIF (25 mg/kg) or high-dose NIF (50 mg/kg) every day. Mice were sacrificed on day 28.
In the therapeutic model, mice were intratracheally administered BLM (~ 1 mg/kg) while the sham-operated group was administered with the same volume of physiological saline. All bronchial drip only once at day 0. On the first day of BLM bronchial drip, the health status of mice was observed, and the previous feeding pattern was maintained until the 13th day after modelling. On the 14th day, all BLM mice were randomly divided into 3 groups. n = 9 (Vehicle), n = 8 (25 mg/kg, 50 mg/kg NIF). The sham operation group (n = 4) and the vehicle group were began intraperitoneally injected with dissolvent (5:35:60 ratio of DMSO: polyethylene glycol 400: normal saline) every day on day 14. At the same time, the NIF groups received intraperitoneal injection of low-dose NIF (25 mg/kg) or high-dose NIF (50 mg/kg) every day. Mice were sacrificed on day 28.
On the 28th day, some mice were used for the extraction of bronchoalveolar lavage fluid (BALF), and the remaining mice were used to collect blood from the eyeballs into EP tubes without coagulant. After standing at 4℃ for almost 24 h, the blood was centrifuged at 3000g/10 min to obtain the serum for ELISA detection.
Extraction of BALF
BALF was extracted according to previous methods reported in the literature  with some modifications. After 28 days of administration, the mice were anaesthetized using 10% chlorine hydrate. Anaesthetized mice were fixed on a clean foam board, and the skin around the neck was sterilized using alcohol-soaked cotton swabs. The skin of the mouse neck was cut, the neck fat was separated, and the mouse bronchi were located. An indwelling needle was inserted into the mouse bronchus and fixed using cotton thread. Then, 1 mL of normal saline was drawn continuously and slowly 3 times through the indwelling needle to the lung, and the final normal saline was collected as BALF. The recovery rate was greater than 80%.
H&E and Masson’s trichrome staining
The collected left lower lung from mice was fixed in 4% paraformaldehyde for one week, dehydrated and embedded in paraffin. The embedded tissue was cut into 3–5 μm thick sections. The tissue sections were stained separately according to the instructions of H&E staining and Masson trichrome staining (BASO, BA-40798). A Panoramic MIDI II 3DHISTECH digital pathology system was used to acquire section images. CaseViewer software was used for processing. Fibrosis was scored as previously described. Fibre volume fractions were determined using ImageJ.
A hydroxyproline determination kit (#A030-2-1, Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China) was used to analyse the hydroxyproline content of lung tissue samples. In brief, tissue was hydrolysed and neutralized using sodium hydroxide. Then, chloramine-T and dimethylaminobenzaldehyde were sequentially added. Finally, a spectrophotometer was used to determine the absorbance at 550 nm to evaluate hydroxyproline content in the lung tissue. Results are expressed as μg/mg wet lung tissue.
Immunohistochemical (IHC) staining
IHC staining was performed on lung tissue sections using an IHC staining kit (ZSGB-BIO Co., Beijing, China) as previously described . Paraffin-embedded lung sections were stained with primary antibodies (α-SMA, Collagen I and p-Stat3 (TY705)) using a DAB Detection Kit (ZSGB-BIO Co., Beijing, China).
Quantification of immune cells in the lung tissue
Lung tissue was disrupted according to a previously described method and lysed with collagenase (37 °C for 90 min) . Changes in the cell ratio were assessed by flow cytometry (BD LSR II) after incubation with different antibodies. The data were analysed using FlowJo software.
Levels of TNF-α, IL-6 and other factors in the BALF were analysed by ELISA using commercially available kits (RAB0477, RAB0308, Merck Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Levels of TNF-α, IL-2 and other factors in murine serum were analysed by ELISA using commercially available kits (#560,484, Cell Signaling Technology Company, MA, USA) according to the manufacturer’s instructions.
The data are presented as the mean ± SD of three independent experiments. Unpaired two-tailed Student’s t tests were used to compare two groups, and one-way ANOVA was performed for multiple group comparisons followed by Tukey’s test. P-values < 0.05 were considered statistically significant; individual P-values are indicated by asterisks in the figures: *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01 and ###P < 0.001. Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad software, San Diego, CA, USA).