Cell culture

Human embryonic lung fibroblasts (WI-38, EK-Bioscience, Shanghai, China) and bone marrow mesenchymal stem cells (MSCs) were cultured in DMEM (L110KJ, EK-Bioscience, Think-Far Technology Co., Ltd., Beijing, China) added with 10% fetal bovine serum (FBS, SH30079.02, Lianshuo Biotech, Shanghai, China) and 1% penicillin/streptomycin (BW41610024, Spectrum Chemical Manufacturing Corp, Shanghai, China) at 37˚C with 5% CO2. Observation of the morphology and growth of BMSCs was performed under a microscope, followed by photographing and preservation.

Cell transfection

The small hairpin RNA of CBLB (sh-CBLB), CBLB overexpression plasmid (pc-CBLB), and MAPK14 overexpression plasmid (oe-MAPK14) were provided by GenePharma (Shanghai, China). WI-38 cells or mesenchymal stem cells (MSCs) were seeded into 12-well plates after trypsinization. Cell transfection was performed when the cell density reached 70–80%. Two EP tubes were taken and filled with Opti-MEM (31985070, Solarbio, Beijing, China), to one of which Lipofectamine 3000 (L3000150, Thermo Fisher, Waltham, MA, USA) was added, and to the other, the plasmid or shRNAs were added. The original medium in the 12-well plates was aspirated, and the mixtures were added to culture plates for transfection. Subsequent experimental treatments were then carried out as required.

Exosome isolation and identification

MSCs cultured in a complete DMEM medium (L110KJ, EK-Bioscience) or MSCs transfected with sh-NC or sh-CBLB were used for exosome isolation through the differential centrifugation method [20]. The isolated pellets were identified by western blotting analysis of exosome marker proteins (CD9, CD81, and TSG101).

The exosome suspension was dropped onto the carbon grid that had been fixed in place. The excess exosome suspension was blotted dry with filter paper. Twenty microliters of 2% phosphotungstic acid (Amresco-0371-500G, Ybiotech, Shanghai, China) were then applied to the carbon grid and left to stand for 20 s. The excess phosphotungstic acid was blotted dry, and the carbon grid was then placed in a glass dish and observed under a transmission electron microscope (TEM, Thermo Fisher). Additionally, the size of MSC exosomes was measured using a nanoparticle tracking analyzer (NTA, Horiba, Shanghai, China).

Cell treatment

WI-38 cells were exposed to a concentration of 10 µg/mL of lipopolysaccharide (SigmaL-2880, LPS, Lianshuo Biotech) for a duration of 24 h to establish an in vitro IP model. This exposure was designed to mimic the inflammatory conditions that might occur in a biological system during IP. Furthermore, a population of WI-38 cells, consisting of 5 × 105 individual cells, was then incubated in the presence of 2 µg of exosomes, followed by LPS stimulation. The purpose of this incubation was to investigate and determine the subsequent effects that these exosomes might have on the gene expression profiles and the overall cellular functions of the LPS-induced WI-38 cells. To determine whether MSC-derived exosomes could be transferred into WI-38 cells, the exosomes were incubated with cell membrane green fluorescent probe DiO (EXOPDiO20-1, Rengen Biosciences, Chenyang, China) at 37 °C for 15 min and then added to WI-38 cells for 48 h, followed by observation under a microscope.

Flow cytometry analysis

The cells from each group were collected and prepared into single-cell suspensions. The cells were incubated with PE-conjugated fluorescent antibodies against CD11b (E-AB-F1081D, Elabscience, Wuhan, China), CD45 (E-AB-F1137D, Elabscience), CD34 (E-AB-F1143D, Elabscience), CD105 (E-AB-F1310D, Elabscience), CD90 (E-AB-F1167D, Elabscience), and CD73 (E-AB-F1242D, Elabscience). Subsequently, the cells were resuspended in PBS (P1020, Solarbio) and analyzed using a flow cytometer.

Oil red O staining

MSCs were seeded into 6-well plates that had been pre-coated with 0.1% gelatin (Amresco-9764-100G, Ybiotech). When the cell confluence reached over 90%, DMEM medium containing dexamethasone (BioVision-1042-10G, Ybiotech), 5 mg/L insulin (PA126938, Think-Far Technology Co., Ltd), 0.5 mmol/L 3-Isobutyl-1-methylxanthine (3-Isobutyl-1-methylxanthine, ybiotech), and 60 µmol/L indomethacin (Cayman-70270-25000, ybiotech) was added to the 6-well plates. After approximately 2 weeks, cell treatment was performed using the Oil Red staining Kit (AY1515, AngYu Biotech, Shanghai, China), and the cells were observed and photographed.

Alkaline phosphatase (ALP) staining

MSCs were induced for osteogenesis in a DMEM medium containing 107 mol/L dexamethasone (BioVision-1042-10G, Ybiotech), 50 µg/L vitamin C (ab75764, Khayal Bio-Technology Co., Ltd., Wuhan, China), and 2 mmol/L β-Glycerophosphate disodium salt pentahydrate (HY-D0886, MedChemExpress, Princeton, NJ, USA) for 14 days. The culture medium in the well plates was removed, and the cells were fixed with 4% paraformaldehyde, followed by processing using the ALP staining kit (CTCC-JD002, PH Biotechnology, Jiangyin, China). After removing the distilled water, the samples were photographed under a microscope.

Western blotting assay

RIPA lysis buffer prepared using phenylmethanesulfonyl fluoride (ST2573, Beyotime, Shanghai, China) and phosphatase inhibitors (P1082, Beyotime) was added to cells or exosomes for protein isolation. The lysates were incubated at 95˚C for 10 min and then subjected to electrophoresis. The protein bands were transferred onto polyvinylidene fluoride membranes. The membranes were incubated with the antibodies against TNF-α (ab183218, 1:1000, Abcam), CD9 (ab236630, 1:1000, Abcam), CD81 (ab109201, 1:5000, Abcam), TSG101 (ab125011, 1:3000, Abcam), Calnexin (ab22595, 1:1000, Abcam), CBLB (ab315021, 1:1000, Abcam), IL-6 (ab233706, 1:1000, Abcam), MAPK14 (ab170099, 1:2000, Abcam), GAPDH (ab9485, 1:2500, Abcam), and IL-1β (ab283818, 1:1000, Abcam). After incubation with the secondary antibody (ab6721, 1:5000, Abcam), the membranes were placed onto filter paper to absorb the moisture and then transferred to the tray of the imaging apparatus. The ECL chemiluminescence kit (P0018M, Beyotime) was used to evenly apply the developing solution onto the bands, and the imaging was performed with the results being photographed for preservation.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Following the instructions of the RNAiso Plus reagent (9108, TaKaRa, Dalian, China), the study isolated RNA. After quantification with a NanoDrop 2000 (Thermo Fisher), RevertAid cDNA synthesis reagents (K1691, Thermo Fisher) were used to obtain cDNA. Real-time fluorescence quantification was carried out utilizing primers (shown in Table 1) and SYBR Green reagent (11762500, Thermo Fisher). Gene expression was analyzed using the 2−ΔΔCt formula.

Table 1 Primer sequences used for qRT-PCRCell counting kit-8 (CCK-8) assay

WI38 cells were treated accordingly based on the different experimental purposes. CCK-8 solution (C0038, Beyotime) was added to each well, ensuring no bubbles were present to prevent the OD readings from being affected. After an incubation period of 1 to 4 h, the absorbance was measured at 450 nm using a microplate reader.

5-Ethynyl-2’-deoxyuridine (EdU) assay

An appropriate number of WI-38 cells were cultured in 96-well plates and incubated overnight. The EdU working solution (ST067, Beyotime) was added to the wells in an equal volume to the culture medium. 4% paraformaldehyde was added to each well for fixation for 15 min. 0.3% Triton X-100 was added for incubation. After aspirating out the washing solution, 4’,6-Diamidino-2-Phenylindole (C1002, Beyotime) was added. Fluorescence detection was performed under a fluorescence microscope.

Cell apoptosis analysis

WI-38 cells were transfected and treated with MSC-derived exosomes. The cells were digested with trypsin and then the digestion was stopped by adding a complete medium containing FBS. These cells were then prepared into a cell suspension and distributed into flow cytometry tubes. ANNEXIN V-FITC/PI apoptosis detection kit (CA1020, Solarbio). Annexin V-FITC (Solarbio) was added to the cell suspension and incubated. The cell pellets were resuspended in PBS buffer, and propidium iodide (PI, Solarbio) staining was added for a 5-minute incubation. The cells were placed in a flow cytometer to detect the fluorescence intensity.

Enzyme-linked immunosorbent assays (ELISAs)

The production of pro-inflammatory cytokines including IL-6, IL-1β, and TNF-α in cell supernatant or serum samples was analyzed using the ELISA kits (PI330, PI326, PI305, PI301, PT518, and PT512, Beyotime). The optical density values at 450 nm wavelength were measured using a microplate reader.

Animal experiment

C57BL/6JNifdc mice (male, 6–8 weeks old) used in this experiment were sourced from Hunan Slyke Jingda Experimental Animal Co., LTD (Changsha, China). All mice were SPF-grade and were modeled in the animal facility of Jinan City People’s Hospital. Twenty C57BL/6 mice were randomly divided into four groups: control (CON), model (LPS), LPS + MSCsh−NCEXO, and LPS + MSCsh−CBLBEXO, with five mice in each group. The mice were then administered a single intratracheal instillation of LPS (SigmaL-2880, 5 mg/kg, Lianshuo Biotech) [21] or physiological saline. Four hours after LPS exposure, the mice were intravenously injected with 50 µL of exosomes extracted from MSCs transfected with sh-NC or sh-CBLB. Twenty-four hours later, the mice were euthanized by intraperitoneal injection of 40 mg/kg pentobarbital sodium (P3761, Lianshuo Biotech) followed by cervical dislocation. Lung tissue and serum samples were collected from the mice. The study was approved by the Animal Care and Use Committee of Jinan City People’s Hospital.

Haematoxylin and Eosin (HE) staining

The assay was performed according to the guidebook of the HE staining kit (G1120, Solarbio). The lung tissues of mice were placed in 4% paraformaldehyde solution for fixation, with a duration of at least 24 h. The sections were sequentially placed in xylene and ethanol solutions. Staining was performed with hematoxylin, followed by counterstaining with bluing reagent, and a 10-second water rinse. After staining with eosin, the sections were sequentially incubated in alcohol, ethanol, and xylene for 5 min each. The sections were observed under a light microscope for lung tissue morphology.

Masson staining assay

The assay was performed using the Masson Stain Kit (60532ES66, Yeasen, Shanghai, China) to visualize collagen and muscle fibers within tissue samples. In brief, the lung tissue sections of model mice were stained in Weigert’s iron hematoxylin solution for 60 s. The sections were then slightly washed with running water and stained with ponceau-fuchsin solution. The sections were differentiated in 1% phosphomolybdic acid solution for 8 min. Aniline blue counterstain solution was added for staining for 5 min. After rinsing with absolute ethanol, the sections were mounted with neutral gum. Microscopic observation and photography were performed.

Co-immunoprecipitation (Co-IP) assay

The assay was performed using the Co-Immunoprecipitation Kit (WLA112a, Wanleibio, Shenyang, China). WI-38 cells were cultured in 6-well plates, and the cells were collected into EP tubes when the density reached approximately 90%. After centrifugation, the cell pellets were lysed with RIPA lysis buffer (P0013B, Beyotime). The mixtures were then centrifuged at 12,500 rpm at 4 °C. Thirty micrograms of protein were taken as a positive control Input. Based on the antibody instructions, the primary antibodies against MAPK14 (ab170099, 1:100, Abcam) and CBLB antibody (A302-902 A, 1:200, Thermo Fisher) as well as IgG antibody were added to the remaining proteins. The following day, Protein A + G Agarose was added, and the mixtures were gently rotated at 4 °C for 4 h. The collected pellets were subjected to SDS-polyacrylamide gel electrophoresis using CBLB (ab315021, 1:1000, Abcam), MAPK14 (ab170099, 1:2000, Abcam), and UB antibody (58395, 1:1000, CST, Boston, MA, USA).

Protein stabilization analysis

WI-38 cells transfected with sh-NC or sh-CBLB for 48 h were digested with trypsin to prepare a cell suspension. The cell suspension was seeded into 24-well plates and placed in an incubator for continued culture. Cycloheximide (50 ng/mL, CHX, 239763-M, Sigma, St. Louis, MO, USA) was added to inhibit protein synthesis, and time points of 0, 3, 6, and 12 h were set for both cell types. The cells were harvested and western blotting analysis was conducted to assess the stability of MAPK14 protein.

Statistical analysis

Experimental data were analyzed statistically using GraphPad Prism software version 8.0. For data that were continuous and normally distributed, the mean ± standard deviation was used to represent the values. Comparison between two groups of data was performed using Student’s t-test, while comparisons among multiple groups were conducted using one-way ANOVA. P < 0.05 was considered statistically significant.