Reagents
The enzyme-linked immunosorbent assay (ELISA) kits in this study were TNF-a (cat #: MEC1003), IL-6(cat #: MEC1008), GM-CSF(cat #: MEC1004), and TGF-b( cat #: MEC1012) were purchased from ANOGEN (Mississauga, Ontario, CANADA); The PAI-1 ELISA kit was purchased from Molecular Innovations (Novi, MI). HYP-detection kit (cat #: A030-1-1) was purchased from Njjcbio, China, Sirius red stain (G1018; Servicebio).
Animals
C57BL/6 mice (12 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (SCXK2018-0003) and raised in a pathogen-free rodent facility according to the procedures approved (2021-01-SYXK-DJC–002; Additional file 1) by the Committee on the Ethics of Animal Experiments of Fudan University. The mice were housed in separate stainless-steel cages (six mice per cage) in a temperature-controlled environment (20-24 °C) on 12 h light-dark cycles with unrestricted access to food and water. All procedures of this study were followed by the “Experimental Animal Ordinance” of Fudan University. After adaptive feeding for one week, during the construction of the animal model with BLM, we used 1% pentobarbital sodium, to anesthetize the mice via intraperitoneal injection 0.15ml/20g, while PET/CT process, after injection of 5MBq [18F]-FDG mice were anesthetized with isoflurane, in an induction chamber, and the anesthesia machine was set to 1.5% isoflurane with a flow rate of 4.5 liters/min oxygen. Observe the reduction of animals’ breathing (the percentage of isoflurane can be adjusted ± 0.5% depending on the susceptibility of the animals to isoflurane anesthesia). For lung function testing we used 1% pentobarbital sodium,via intraperitoneal injection of 0.15ml/20g to the mice and we used a surgery board during tracheotomy (at approximately 70° from horizontal)[20]. After 1 week of adaptive feeding, the mice were randomly divided into nine groups, saline intraperitoneal (100ml) control group, IPC, intraperitoneal injection of BLM low dose (20mg/kg) group, IPL, high dose(50mg/kg) group, IPH, saline intratracheal administration (50ml) control group, ITC, intratracheal administration of BLM low dose (3mg/kg) group, ITL, high dose (5mg/kg) group, ITH, saline tail vein injection(100ml) control group, IVC, intravenous administration (tail vein) of BLM low dose(10mg/kg) group, IVL, high dose (20mg/kg) group, IVH. For intraperitoneal BLM group, after senitized with 75% of alchohol, we injected 20 mg/kg and 50mg/kg for low dose(IPL) and high dose group(IPH), respectively, with constant volume of 100ml, 100ml saline for control repeated 7days. For intratracheal administration of BLM, nonsurgical transoral instillation of BLM into mice lung method was used[21]. 2% sodium pentobarbital was prepared for anesthesia and injected into the abdominal cavity according to the corresponding dose of the mice's body weight(50mg/kg). After anesthesia, mice lied supine on a fixed table with the limbs fixed, and the neck is shaved. After disinfection, laryngoscopes and LED light was used to make sure the BLM could accurately purfused into trachea.In the low(ITL) and high dose(ITH) group , mice were injected with 3mg/kg, 5 mg/kg BLM dissolved in 50ml of saline,respctively, while in the control group mice were injected with an equal volume of saline. Tail vein injection BLM mouse model produced as followed: Put the mouse into a mouse fixer [22] (mouse injection cone with restrainer and LED GLOBALEBIO GEGD-Q9G), expose the tail, disinfect with 75% alcohol, and expose the tail vein. In the low dose(IVL) and high dose(IVH) group, mice were given BLM injection at 10mg/kg and 20 mg/kg, respectively, with a 1mL syringe (BLM was dissolved with normal saline at a concentration of 2 mg/mL) for 7 days; In the saline control group, mice were injected with 5 mL/kg normal saline through the tail vein for 7 days.
Survival status and body quality testing
During the experiment, the activity, changes in the body weight, eating, drinking, fur, and survival status of the mice in each group were observed.
PET/CT imaging and analysis
At day 25-26 animals were received successive PET/CT labelled with [18F] -FDG [11] after anesthetized through isoflurane (1.5%) inhalation for intraperitoneal injection of 5MBq of 18F-FDG PET/CT 20 min before imaging. Mice were then maintained under anesthesia (1.5%) and placed on an imaging heated bed inside an Inveon MM PET (SIEMENS, JAPAN).
Lung function test in mice
All the mice were anesthetized with an intraperitoneal injection of 1% pentobarbital (0.15ml/20g), and tracheostomy was conducted, as previously described [23], with a standard catheter. All the mice were tracheostomized and placed in a forced pulmonary function testing (PFT) system (Buxco, NY, USA) with FinePointe and FinePoint Control Panel software in the supine position on the bed within the plethysmograph, which simulated clinical pulmonary testing routinely performed on humans.
The indexes related to inflammation, fibrosis in BALF, serum, and lung tissue were determined by ELISA
The enzyme-linked immunosorbent assay (ELISA) kits in this study determined TGF-b1, TNF-a, IL-6 and GM-CSF antigen in BALF and Serum. The PAI-1 and HYP antigen in lung tissue were determined by using their ELISA kit.
Histopathological tests
Histopathological Examination For histological analysis, pulmonary tissues were fixed with 4% paraformaldehyde and embedded in paraffin. The paraffin blocks were cut at 5 μm using a microtome. Sections stained against Masson Trichrome [15]. Collagen deposition of the sections were observed and assessed with an optical microscope and quantified with Image-Pro Plus6.0 software. PSR staining and quantification[24-26] were used in this test. The tissue sections were deparaffinized and rehydrated in a graded ethanol series and then incubated for 1 h in PSR staining. The stained sections were analyzed by using an ECLIPSE Ci-L/Ci-E Clinical Upright Microscope (Nikon; magnification, x200) with a linear polarizer. To avoid missing any details, the filter was tilted to an angle between 0 to 90 until the birefringence became evident and the background became completely black. The focus was then corrected once more [25]. The halogen lamp intensity and exposure time were constant within each image. Under polarized light, Collagen type 1(also known as collagen alpha, Col1A1, and alpha-1 type 1 collagen, Col 1) appeared to red or yellow with strong birefringence, while Collagen type 3 (Col 3) was green with weak birefringence. The areas of Col 1 and 3 staining were analyzed by using Image Pro Plus 6.0 (Media Cybernetics, Inc.).
Transmission electron microscopy images
Blocks were rinsed overnight in 0.1 M phosphate buffer (350 mOsm, pH 7.4) and fixed for two hours in osmium tetroxide (1% osmium tetroxide in 0.125 sodium cacodylate buffer, 400 mOsm, pH 7.4). Then the samples were passed through stepwise dehydration in increasing concentrations of ethanol (50–100%), rinsed with propylene oxide, and embedded in Araldite. Blocks were then cut into ultrathin sections (50-70 nm) and contrast stained with saturated uranyl acetate and bismuth subnitrate. Sections were examined at an accelerating voltage of 60 kV 80 by using a Zeiss EM 10 C transformation electron microscope (HITACHI H-7500). Micrographs of a carbon grating replica were taken for calibration[27, 28].
Statistical analysis
GraphPad Prism 8.0 statistical software was used for statistical processing of the related experimental data, all results flowed x±SD. Normality tests and homogeneity of variance tests were conducted for the measurement data of each group. One-way ANOVA was used for the data in line with normal distribution and homogeneity of variance, and the Tukey test was used for pairwise comparison between multiple groups. P <0.05 was considered statistically significant(*p<0.05, **p<0.01, ***p<0.001,****p<0.0001).