Regents
Phosphate-buffered saline (PBS), penicillin-streptomycin solution, trypsin, eosin (E8090) and neutral resin (G8590) were from Beijing Solarbio Science & Technology Co., Ltd. Glutaraldehyde, acetone, xylene and absolute ethanol were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. Hematoxylin (G1004) was from Wuhan Servicebio Technology Co., Ltd.
MSCs cell culture
Umbilical cords were dissected from 16-day pregnant female SD rats in a sterile condition, sliced into 1 mm2 pieces, and washed with PBS. The tissues were digested with 0.25% trypsin for 30 minutes and inoculated in DMEM/F12 medium (Hyclone, SH30023.01) containing 10% Fetal Bovine Serum (Gibco, 10270-106) and 1% Penicillin-Streptomycin. The cells were cultured in a 5% CO2 incubator at 37 ℃.
MSCs cell identification
We collected 1×106 MSCs and incubated cells in the dark with 2 µL CD44-FITC (eBioscience™, 11-0860-81) and CD45-PE (eBioscience™, 12-0461-80) antibodies. Then, we washed the cells with PBS, centrifugated the cells and aspirated the supernatant. Finally, cells were resuspended and identified by Flow cytometry.
MSCs transfection and exosome collection
The MSCs in the normal control (NC) group were non-transfected. And the MSCs in the mimics NC group and inhibitor NC group were transfected with empty vectors, while the OV-Let-7d group was transfected with miRNA Let-7d mimics (5’-AGAGGUAG UAGGUUGCAUAGUU-3’) and in the si-Let-7d group was transfected with miRNA Let-7d inhibitors (5’-UCUCCAUC AUCCAACGUAUCAA-3’). All transfection vectors were labeled with fluorescence and observed by immunofluorescence microscopy to verify the transfection efficiency. After MSCs’ transfections were processed for 48 hours, we centrifugated cells at 2000g for 20 minutes and collected exosomes from the supernatant. The exosomes were centrifugated at 10000g, 4 ℃ for 1 hour and resuspended with PBS.
The miRNA Let-7d mimics and inhibitors were synthesized by GenePharma (Shanghai, China).
Mice models construction and exosome intervention
The 30 BALB/c male mice aged 6 to 8 weeks were from China Three Gorges University and fed by Wuhan Myhalic Biotechnology Co. Ltd. After we anesthetized the mice with isoflurane, the skin was dissected and the trachea was separated. Subsequently, we injected 0.2 mL bleomycin (4 mg/L) or 0.2 mL saline into the trachea. Immediately, we rotated the mice to distribute the drug evenly, then sutured and sterilized the skin.
The mice in the various groups were respectively injected with OV-Let-7d exosome (3 mg/kg), si-Let-7d exosome (3 mg/kg), or saline through the tail vein for 3 days. Lastly, we injected 100 mg/kg sodium pentobarbital into the mice intraperitoneally to anesthetize and capture the eye blood and lung tissues.
Transmission Electron Microscope Scan
The suspension of MSCs’ exosomes was dropped on the copper nets and stood for 4 to 5 minutes. Then the nets were clamped out and washed with double-distilled water. The nets were stained with uranyl acetate for 5 minutes in the dark and washed again, and dried with filter papers. We scanned their morphology through a transmission electron microscope (H-7700, Hitachi, Ltd.).
Western blot
Lung tissues were homogenized in RIPA lysate (R0010, Beijing Solarbio Science & Technology Co., Ltd) containing 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 1 mM protease inhibitor on ice for 20 minutes. We determined the protein concentration by BCA Protein Assay Kit (P0009, Beyotime, Beijing, China) after centrifugation. Then the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was added and the proteins were boiled at 95 ℃ for 10 minutes. 30 µg total proteins were separated by SDS-PAGE and transferred to the polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore, Merck KGaA, Darmstadt, Germany). The membranes were blocked in 5% skim milk for 1 hour at room temperature, washed thrice for 10 minutes with Tris-buffered saline with Tween-20 (TBST), and incubated with primary antibodies at 4 ℃ overnight. Then we washed the membranes three times for 10 minutes and incubated with an HPR-labeled goat anti-rabbit IgG second antibody (SAB43714, Bioswamp, 1:20000) for 1 hour at 37 ℃. The protein expression was detected by the Gel Imaging System and calculated by ImageJ. The primary antibodies against CD63 (PAB33929), Col1a1 (PAB44576), Col3a1 (PAB43825), FN (PAB31274), E-cadherin (PAB43792), N-cadherin (PAB30130), Snail (PAB33921), Slug (PAB30534) and GAPDH (PAB36269) were from Bioswamp (Wuhan, China).
Hematoxylin and eosin (H-E) staining
We embedded mice lung tissues in paraffin and sliced them to 3 µm. Then the slices were flattened in water and baked to dryness. Before immunostaining, 3-µm thick lung tissue sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, and washed in PBS, and then stained with hematoxylin and eosin. After staining, sections were dehydrated through increasing concentrations of ethanol and xylene.
Immunohistochemical (IHC) staining
Antigens were unmasked by microwaving sections in 10 mmol/L citrate buffer, pH 6.0 (15 minutes). The sections were treated with 5% H2O2 for 10 min and blocked in 10% goat serum for 25 min. The immunostaining was undertaken using the avidin-biotinylated enzyme complex method with antibodies against α-SMA (PAB30319, Bioswamp, China) at a concentration of 1 ug/ml, PDGF-A (ab203491, Abcam, UK) at a concentration of 1 ug/ml, and equivalent concentrations of polyclonal nonimmune IgG controls. After incubation with the MaxVisionTM HRP-Polymer anti-Mouse/Rabbit IHC secondary antibody (Maixin, Biotechnology Development Co. LTD, China) and subsequently with streptavidin solution, color development was performed using 3,3 -diaminobenzidinetetrahydrochloride (Vector Laboratories) as a chromogen. Sections were counterstained using Gill-2 hematoxylin (Thermo-Shandon, Pittsburgh, PA).
Masson staining
The slides were stained with hematoxylin for 5 min and ethanol was differentiated with hydrochloric acid. We stained the slides with ammonia, acid fuchsin and aniline blue. Then, the slides were dehydrated with ethanol for 15 seconds and soaked with xylene for 5 minutes. The neutral resin was attached to the slides, and photos were taken. All stains were obtained from the Masson kit (G1340, Solarbio, Beijing, China).
Enzyme-linked immunosorbent assay (ELISA)
Blood was collected from the orbits of mice and the serum was drawn after centrifuged. The con contraption of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Hyaluronic acid (HA), and transforming growth factor-β (TGF-β) were detected by ELISA kits (Wuhan Gene Beauty Biotechnology Co., Ltd., China).
Statistical Analysis
GraphPad Prism 8.0 software was used for the statistical analysis, and all data are presented as mean ± standard deviation (SD). One-way ANOVA anaylisis was used for the comparation between more than two independent groups. P < 0.05 was considered that as the statistically significant.