Establishment of pulmonary fibrosis model induced by silica. Pulmonary fibrosis was induced in mice (C57BL/6 background) by intratracheal instillation with 50mg/kg of silica particles (size distribution: 99% between 0.5 and 10µm, 80% between 1 and 5µm, average particle diameter 1.7µm, obtained from SigmaAldrich, St. Louis, MO, USA) dissolved in 0.05 ml sterile saline. Mice administered the same volume of sterile saline served as controls. On days 8, 15 and 22 following the silica treatment, mice were intravenously injected with Scrambled siRNA or NPM1 siRNA-loaded liposomes. Finally, the mice were euthanized on days 28 after the silica treatment. The lung tissues were isolated and stored at – 80°C for further analysis.
Establishment of pulmonary fibrosis model induced by BLM. Pulmonary fibrosis was induced in mice (C57BL/6 background) by intratracheal instillation with 6 mg/kg BLM (Warbio, Nanjing, China) in 40 µl of sterile saline. Mice administered the same volume of sterile saline served as control subjects. Finally, the mice were euthanized on days 7, 14 and 21 following the BLM instillation, respectively. The lung tissues were isolated and stored at – 80°C for further analysis.
Pathological staining and histopathologic assessment. The mice lung tissues were harvested at 28 days after silica or saline administration, fixed with formalin solution overnight and embedded in paraffin. The tissue sections were then stained with hematoxylin and eosin (H&E), Masson’s trichrome stain and Sirius red.
Hydroxyproline assay. The amounts of lung collagen deposition were assessed by measuring the hydroxyproline content of lung tissue sections with a hydroxyproline assay kit (Jiancheng Bioengineering Institute, Nanjing, China). In accordance with the manufacturer’s protocol, the samples were determined by the spectrophotometer at 550 nm. The hydroxyproline concentration in the sample was calculated from a standard curve and related to the amount of lung tissue used. The hydroxyproline contents in the lung tissues are given as µg of hydroxyproline per mg of lung tissue.
Cell culture and treatment. The human lung epithelial cells (BEAS-2B), human lung fibroblast (MRC-5) cells were commercially obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). BEAS-2B cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies/Gibco, Grand Island, NY, USA), and the MRC-5 cells were maintained in Minimum Essential Medium (MEM, Life Technologies/Gibco, Grand Island, NY, USA). Primary mouse pulmonary fibroblasts (PMLFs) were isolated from the lung tissues of C57BL/6 mice. Briefly, fibroblasts were generated by mincing lung tissue into submillimeter-sized pieces, plated evenly in 60mm plates containing 4 ml of medium, which was changed after 24 h. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies/Gibco, Grand Island, NY, USA).
All of the culture media were containing 10% fetal bovine serum (Biological Industries) and antibiotics (penicillin and streptomycin, Life Technologies/Gibco, Gaithersburg, MD). Cells were cultured at 37°C in a 5% CO2 atmosphere. Epithelial cells were treated with 0, 50, 100, 150, 200, 250µg/ml Silicon dioxide (SiO2) (Sigma-Aldrich, St. Louis, MO, USA); MRC-5 and PMLFs cells were treated with 0, 1, 2, 5 ng/ml exogenous IL33 (PeproTech) for 24h.
Cell transfection. Small interfering RNAs (siRNAs) specific for IL33, ST2, c-Jun, NPM1 were synthesized by Generay Biotech (Shanghai, China). Cells were transiently transfected using riboFECT CP Regent (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. Transfection efficiency was monitored by RT-PCR at 48 h after transfection. The sequence of human IL33: sense: GCUCUGGCCUUAUGAUAAATT; antisense: UUUAUCAUAAGGCCAGAGCTT.
The sequence of human ST2: sense: GGAUUGGUAUUACUCACAATT; antisense: UUGUGAGUAAUAACCAAUCCTT. The sequence of mouse ST2: sense: GGAAUGGUAUUACUCAGAUTT; antisense: AUCUGAGUAAUACCAUUCCTT. The sequence of human c-Jun: sense: GAAAGUCAUGAACCACGUUTT; antisense: AACGUGGUUCAUGACUUUCTT. The sequence of mouse c-Jun: sense: GCAAAGAUGGAAACGACCUTT; antisense: AGGUCGUUUCCAUCUUUGCTT. The sequence of human NPM1: sense: GAGCACCAGUUAUCUUUAATT; antisense: UUAAAGAUAACUGGUGCUCTT. The sequence of mouse NPM1 (#1): sense: CUAUCACUUUAAAGUGGAUTT; antisense: AUCCACUUUAAAGUGAUAGTT. The sequence of mouse NPM1 (#2): sense: CAGAAGCAAUGAACUAUGATT; antisense: UCAUAGUUAUUGCUUCUGTT. The sequence of mouse NPM1 (#3): sense: GACAGCAUCUAGUAGCUGUTT; antisense: ACAGCUACUAGAUGCUGUCTT.
c-Jun-plasmid and a vector plasmid were purchased from Generay Biotech (Shanghai, China), and 2µg of purified DNA was mixed with transfection reagent and applied to the cells. 48 h after transfection and co-transfection with IL33, the cells were analyzed by Western blot and RT-PCR.
Real-time PCR. Total RNA from collected tissues or cells was extracted using the Trizol reagent (Tiangen Biotech, Beijing, China). RNA quality and concentration were measured by Nanodrop 2000 spectrophotometer (Termo, Waltham, MA). All mRNA was detected using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co, Nanjing, China) in the ABI 7900HT Real-Time PCR system (Applied Biosystems). Fold changes in the expression levels were calculated using the 2−∆∆Ct method and normalized using GAPDH as the endogenous control.
Western blot and antibodies. For western blot assay, all cells were washed twice times with PBS and then used with RIPA lysis buffer and phenylmethylsulfonyl fuoride for extraction of total proteins (Beyotime Institute of Biotechnology, Shanghai, China; PMSF, Sigma-Aldrich, St. Louis, MO, USA). The total protein of the mouse lung tissues was extracted with T-PER Tissue Protein Extraction Reagent (Termo Scientifc). Protein concentrations were measured by BCA Protein Assay (Beyotime Institute of Biotechnology, Shanghai, China). A total of 80 µg of protein extracts were separated via SDS-PAGE and transferred onto polyvinylidene difuoride (PVDF) membranes (ISEQ00010, 0.2 µm, Immobilon). Then the membranes were incubated overnight at 4°C with appropriate primary antibodies and appropriate secondary antibodies. Protein bands were visualized using the ChemiDocXRS + imaging system (Bio-Rad Laboratories, Inc). The gray values were analyzed with ImageJ software.
The primary antibodies used were anti-Fibronectin (Abcam); anti-Collagen I (ABclonal); anti-αSMA (Abcam); p-ERK (Cell Signaling Technology); ERK (Cell Signaling Technology); p-c-Jun (Cell Signaling Technology); c-Jun (Cell Signaling Technology); NPM1 (ABclonal); anti-GAPDH (ABclonal).
Cell proliferation assay. PMLFs were cultured in 96-well plates at a density of 2 × 103 cells/well. Cell proliferation was then measured using the EdU proliferation assay (Ribobio, Guangzhou, China), according to the manufacturers’ instructions. Briefly, 18 h after being seeded in the plates, cells were labeled with EdU for 2h at 37°C, treated with 100µL of Apollo reaction cocktail and stained with 100µL of Hoechst 33342. Finally, the cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan).
Cell viability assay. Cell viability was detected with a Cell Counting Kit-8 assay (CCK8, Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. The cells were plated in a 96-well plate, followed by exposure to different treatments for the indicated times. Then 10µl CCK8 reagents were diluted in each well for 1 h at 37°C in 5% CO2, and the 96-well plate was measured at 450 nm carried out with a microplate reader (TECAN Infnite M200, Mannedorf, Switzerland).
Woundhealing assay. Cells were seeded in 6-well plates and cultured until the cells reached 70–80% confluence. Wounds were scratching with a sterile 200µl pipette tip across the monolayered cells to create a straight linear scratch, and a wound gap was performed by a microscope. After indicated treatment, the widths of the wound were followed by the previously described procedure. The wound gap was quantitatively evaluated with ImageJ software.
Immunostaining assay. Cryosections of lung tissues from mice with the onset of silica-induced pulmonary fibrosis were used for immuno-fluorescence staining. The primary antibodies used for staining were as follows anti-αSMA (Abcam), anti-Collagen I (ABclonal), NPM1 (ABclonal), and Cy3-conjugated or FITC-conjugated goat anti-rabbit antibody (Beyotime Institute of Biotechnology, Shanghai, China) were used as fluorescent secondary antibodies. Besides, after the indicated treatment, MRC-5 cells were washed fixed with 4% methanol for 30 min, then stained with anti-αSMA (Abcam) at 4°C overnight and incubated with Cy3-conjugated goat anti-rabbit antibody (Beyotime Institute of Biotechnology, Shanghai, China) for 1h. The DAPI was used to stain the nucleus in cells for 5 min. All the images were acquired with the fluorescence microscope (Olympus, Tokyo, Japan).
ChIP assay. The ChIP assay was performed to analyze the binding of c-Jun to the NPM1 promoter using a ChIP Assay Kit (CCK8, Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, protein and DNA complex was precipitated with specific antibody against c-Jun (Cell Signaling Technology) and immunoglobulin G control (Millipore). Then, the purified DNA was suspended in Tris-EDTA buffer. PCR was performed to analyze the NPM1 promoter region.
Liposomes: Liposomes were raised as carriers to encapsulate siRNA. The lipid mixture including lipidoid, cholesterol, DSPC and mPEG-DMG dissolved in ethanol at a molar ratio of 50:38.5:10:1.5 were prepared. The unentrapped free siRNA was removed by ultrafiltration centrifugation. The hydrodynamic diameter, zeta potential and stability of the liposomes were measured by dynamic light scattering (DLS) (Malvern Zetasizer Nano-ZS, UK). After staining with 2% phosphotungstic acid, the liposomes were characterized by transmission electron microscopy (TEM, Jeol, Japan).
In vivo and ex vivo biodistribution of the liposomes. In vivo biodistribution of the liposomes, after si-NPM1 liposomes were constructed, the prepared liposomes were intravenous injection into the mice after 7 days of silica induction. Subsequently, the mice were anesthetized and photographed at different time points (0h, 24h, 48h) by an in vivo imaging system (IVIS Lumina XR, SI Imaging, AZ, USA). Similarly, the mice were euthanized and the organs were harvested for ex vivo fluorescence imaging, respectively.
Statistical analysis. All experiments were performed in triplicates calculated by GraphPad Prism (San Diego, CA, USA). Statistical analysis was performed using Student’s t-test (between two groups) or one-way analyses of variance followed by Tukey’s multiple comparisons test (more than two groups). Data were expressed as mean ± SD. P < 0.05 was considered statistically significant.