Isolation and identification of hucMSC-Exos
The exosome-specific MSC medium (Yumeibo Biotechnology Co., Ltd., Shanghai, China) was utilized to culture third-generation hucMSCs (Han’s United Bio-Technology Co., Ltd., Beijing, China) until a confluence rate of 80–90% was attained. Informed consent was obtained in accordance with the Declaration of Helsinki and the Medical Ethics Committee of the North China University of Technology (Approval No. 2023016). Subsequently, the culture supernatant was collected and subjected to two rounds of centrifugation: the first at 1000×g for 10 min at 4 °C, followed by a second round at 10,000×g for 10 min at 4 °C to remove dead cells and cellular debris. Following centrifugation, the supernatant was cautiously collected. Exosomes were subsequently isolated via differential centrifugation and stored at − 80 °C for future applications.
Transmission electron microscopy
2 mL of PBS was added to the centrifuge tube in which the exosomes were isolated and mixed well, and 10 µL of the exosome dilution was taken according to a 1:50-fold dilution and added dropwise onto a 2 mm carbon-containing sample-carrying copper mesh, which was left to stand at room temperature for 5 min and then gently aspirated off the excess liquid with a piece of filter paper, negatively stained with a 3% phosphotungstic acid sodium solution (pH6.8) at room temperature for 5 min, and gently washed with double-distilled water for one time and then allowed to air dry at room temperature, and a transmission electron microscope (Hitachi, Tokyo, Japan) to obtain a transmission electron image.
Nanoparticle tracking analysis
To quantify the particle size distribution of exosomes derived from hucMSC-Exos, a NanoSight instrument (ZetaVIEW, Germany) was employed. The collected exosomes were diluted to a concentration of 1 × 106 particles/mL in PBS and subsequently injected into the NanoSight sample chamber using a 1 mL syringe. A laser beam was directed through the exosome particles within the sample chamber, enabling the visualization of the particles via a microscope equipped with a camera, which recorded the Brownian motion of the exosomes for 60 s. This process was repeated three times to ensure reproducibility. The concentration and size distribution of the exosomes were calculated from their motion using Einstein’s equation. To ensure the accuracy of the results, PBS was evaluated prior to the experiment to confirm the absence of particulate contaminants.
Western blot detection of membrane surface markers of exosomes
HucMSC-Exos samples were subjected to SDS-PAGE using 10% polyacrylamide gels. Protein quantification and SDS-PAGE gel electrophoresis were performed according to our previously published protocol [19]. The primary antibodies utilized in this study comprised CD9 (1:1000, ab236630), CD81 (1:1000, ab109201), TSG101 (1:20,000, ab125011), and Calnexin (1:10,000, ab2259), all sourced from Abcam (Abcam, USA). The secondary antibody employed was HRP-goat anti-rabbit IgG (1:10,000, ab6728), which was also sourced from Abcam (Abcam, USA). ECL detection reagents (Ripart Biotechnology Co., Ltd., Hebei, China) were employed for signal detection and imaging. Protein blots were visualized and quantified utilizing ImageJ software.
Animals models and treatments protocols
40 male C57BL/6 mice, each Weighing between 20 and 23 g, were obtained from the Laboratory Animal Center of North China University of Science and Technology. The animals were acclimatized under standard laboratory conditions, including a ventilated room maintained at 25 ± 1 °C, 50 ± 5% humidity, and a 12-hour light/dark cycle. They had unrestricted access to standard food and water. The Laboratory Animal License number is SCXK(X)2019-0008. All procedures adhered to the ARRIVE2.0 guidelines and were approved by the Committee for the Protection and Use of Laboratory Animals at North China University of Technology (Approval No. SQ20230060).
In this study, 40 male C57BL/6 mice were randomly divided into 8 groups (5 mice per group): control groups for 28 days and 56 days, a model group, a group treated with hucMSCs, and a group treated with hucMSC-Exos (the specific grouping details are provided in the Supplementary Materials). After a 5-day acclimatization period, the model was induced under anesthesia using an intraperitoneal injection of 0.3% pentobarbital sodium (40 mg/kg) (Kino Chemical Co, Wuhan, China). Mice in the three experimental groups, excluding the control group, received a single, non-exposed tracheal instillation of 50 µL of silica suspension at a mass concentration of 200 mg/mL. In the exosome group, hucMSC-Exos were administered via caudal vein injection at a concentration of 5 × 1010 particle/mL (100 µL/each) on days 1, 5, and 9 post-silica exposure [18]. In the stem cell group, hucMSCs were administered via tail vein injection at a concentration of 1 × 106 cells/mL (100 µL/each) on days 1, 8, and 15 post-silica exposure [20]. The mice were anesthetized with a 0.3% sodium pentobarbital (40 mg/kg) by intraperitoneal injection on days 28 and 56 after silica exposure. Venous blood was collected via orbital puncture, and subsequently euthanized by cervical dislocation. Lung tissue was then taken from each group of mice (all were left lung tissues of mice), weighed and photographed, and the samples were stored at − 80 °C or fixed for subsequent experiments. No specific exclusion criteria were established, and no mice were excluded from the study. All outcome assessments were conducted by experimenters who were blinded to the group assignments.
General conditions of mice in each group
40 mice were Weighed immediately after grouping and subsequently treated according to the specific requirements of each group. The body Weight of the mice in each group was measured and recorded weekly. On the 28th and 56th days of treatment, lung tissues from the mice in each group were harvested, photographed, and weighed to calculate the lung coefficient. All data were subjected to statistical analysis.
Measurement of oxygen saturation, heart rate and respiration in mice
Arterial oxygen saturation, heart rate, and respiration were measured in mice using the MouseOx system (STARR Life Sciences, Yuyan Scientific Instruments Co., Ltd., Shanghai, China). Prior to measurement, the fur around the neck of the mice was carefully removed, and the dehaired area was secured using a CollarClip sensor to assess arterial oxygen saturation (SpO₂), respiration rate (R), and heart rate (HR). Measurements were recorded for a duration of 5 min following the acclimatization period of the mice and were subsequently averaged over this measurement interval.
Micro CT imaging
A NEMO-II NMC-200 panoramic tomography system (Pingsheng Medical Technology Co., Ltd., Kunshan, China) was employed to obtain high-resolution CT images of mouse lungs, following the manufacturer’s operational guidelines. To maintain the mice in a prone position during imaging, isoflurane was administered for anesthesia. The acquired images were subsequently processed using AVATAR 1.5.0 software to construct three-dimensional representations of the lung tissue.
H&E staining and Masson staining
After formaldehyde fixation of mouse lung tissues, paraffin-embedded sections were prepared. Hematoxylin and eosin (H&E) staining was employed to assess the structural integrity of alveoli and the inflammatory response in the lungs, while Masson’s trichrome staining was utilized to evaluate collagen deposition in the lung tissues. Quantitative analysis was conducted using ImageJ software to measure the area occupied by elastic and collagen fibers.
Western blot analysis of mouse lung proteins
Total proteins were isolated and extracted from mouse lung tissues using RIPA buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China). Protein quantification and SDS-PAGE gel electrophoresis were conducted in accordance with our previously published protocol [19]. The internal reference antibody was β-actin. The following primary antibodies were used: TNF-α (1:500, AF7014), NF-κB (1:1000, AF5006), IL-1β (1:1000, AF4006), IL-6 (1:1000, DF6087), α-SMA (1:800, AF1032), and TGF-β (1:1000, AF1027), all purchased from Affinity Biosciences (Affinity, Jiangsu, China). ECL detection reagents (Ripart Biotechnology Co., Ltd., Hebei, China) were used to detect and image the signals. Protein blots were visualized and quantified using ImageJ software.
Determination of serum tumour necrosis factor-alpha (TNF-alpha) by ELISA kit
Serum levels of TNF-α were measured on days 28 and 56 post-modeling (with 5 mice per group at each time point) to assess the effects of hucMSCs and hucMSC-Exos on lung inflammation in mice. Blood samples were collected, incubated overnight at 4 °C, and then centrifuged at 2500×g. The resulting serum was used for ELISA to quantify TNF-α levels. TNF-α concentrations in mouse serum samples were determined using the Mouse Tumor Necrosis Factor-α (TNF-α) ELISA Assay Kit (ABclonal, Wuhan, China). Following the operational procedures outlined in the instruction manual, the termination solution was added, and the optical density (OD) value was measured at 450 nm within 5 min, using a correction wavelength of 570 nm. A standard curve was constructed to facilitate the calculation of TNF-α concentrations in the mouse serum samples.
Mouse lung tissue hydroxyproline assay
Lung tissue hydroxyproline (HYP) levels were measured using a conventional hydroxyproline assay kit based on the alkaline hydrolysis method (Jiancheng Bioengineering Institute, Nanjing, China). Protein concentrations in lung tissue were Quantified at an absorbance of 550 nm, following the manufacturer’s protocol, and all data were reported in µg/mg of tissue.
High-throughput transcriptome sequencing of lung tissue
High-throughput transcriptome sequencing services were provided by Shanghai Baiqu Biomedical Technology Co., Ltd. Mouse lung tissue specimens were collected from four groups: the 28-day control group, the 28-day model group, the 28-day exosome treatment group, and the 28-day stem cell treatment group. These specimens were immediately frozen in liquid nitrogen. Total RNA was extracted using Trizol reagent (Thermo Fisher, 15596018). The Quantity and purity of the total RNA were assessed using the Bioanalyzer 2100 and the RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511). Only high-quality RNA samples with a RIN number greater than 7.0 were utilized for constructing sequencing libraries. Following RNA extraction, mRNA was purified from 5 µg of total RNA using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) through two rounds of purification. High-quality sequencing data were obtained, and the resulting data were aligned with the reference genome of the species studied. Subsequent analyses, including gene expression quantification, Gene Set Enrichment Analysis (GSEA), differential gene expression analysis, and enrichment analysis, were performed.
Staining of lung tissue for phalloidin
After deparaffinization and washing of paraffin-embedded lung tissues with PBS three times, the tissues were subjected to fixation and permeabilization. Subsequently, 300 µL of TRITC-labeled phalloidin working solution (Cayman Chemical Company, USA) was added and incubated for 30 min at room temperature in the dark. Following incubation, the tissues were stained and sealed using an anti-fluorescence quenching blocking agent containing DAPI (Solaibao Technology Co., Ltd., Beijing, China). Finally, observations and photographs were taken using a fluorescence microscope.
Cell culture
Human monocytes THP-1 (Ripart Biotechnology Co., Ltd., Hebei, China) were cultured in an incubator at 37 °C, 5% CO2 in (medium containing 89% RPMI1640 basal medium, 10% fetal bovine serum superior, 1% dual antibody, 0.1% 0.05 mM2-mercaptoethanol) (Basic Medium Science and Technology Co., Ltd., Shanghai, China), and the cells were cultured when the cell fusion reached 80%–90% for passaging. The cell lines have been identified and tested, and the report shows that they are qualified. Subsequently, THP-1 cells were inoculated into 24-well culture plates at a concentration of 5 × 105 cells/mL and stimulated with phorbol 12-myristate 13-acetate (PMA) at a concentration of 80 ng/mL. After 72 h of induction, THP-1 cells differentiated into macrophages.
Cell grouping and intervention
Differentiated THP-1 cells were categorized into three groups: the blank control group, the SiO₂ model group, and hucMSC-Exos + SiO₂ group. Both the SiO₂ model group and the hucMSC-Exos + SiO₂ group, with a cell concentration of 5 × 105 cells/mL, were treated with a SiO2 suspension (100 µg/mL, Min-U-Sil 5, Sigma-Aldrich, St. Louis, MO, USA; particle size distribution: 97% < 5 μm diameter, 80% < 3 μm diameter, with a median diameter of 1.4 μm) and incubated for 24 h [21]. The hucMSC-Exos + SiO2 group received exosomes at a concentration of 103 particles per cell in conjunction with silica treatment.
HucMSC-Exos uptake by SiO2-stimulated THP-1 cells
To assess the uptake of hucMSC-Exos by THP-1 cells, we labeled hucMSC-Exos with Dir red fluorescent dye (Yumeibo Biotechnology Co., Ltd., Shanghai, China), the cell membranes of THP-1 cells with Dio green fluorescent dye (Ripat Biotechnology Co., Ltd., Hebei, China), and the nuclei of THP-1 cells with Hoechst blue fluorescent dye (Biyuntian Biotechnology Co., Ltd., Shanghai, China) for observation. Prior to labeling, THP-1 cells were treated with a 100 µg/mL SiO₂ suspension. Subsequently, the labeled hucMSC-Exos were co-incubated with the treated THP-1 cells, and the cellular uptake of hucMSC-Exos was evaluated using fluorescence microscopy.
Staining of THP-1 cells with phalloidin
Cells were cultured in 24-well plates with cell crawlers. After 72 h of PMA stimulation, THP-1 cells were further cultured until their density reached 50%−60%. The cells were then washed twice with 1×PBS (pH7.4), which was pre-warmed to 37 °C. Next, the cells were fixed in 4% paraformaldehyde at room temperature for 10–30 min. After fixation, the cells were washed 2–3 times with PBS, with each wash lasting 10 min at room temperature. The freshly prepared working solution of phalloidin was used to completely cover the cells, which were stained for 20–90 min at room temperature in the dark. Following staining, the cells were washed three times with PBS, with each wash lasting 5 min. Finally, the coverslips were removed from the cell crawlers, sealed with a mounting medium containing DAPI, and observed under a fluorescence microscope.
Immunofluorescence staining
THP-1 cells were fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.5% Tween20 for 20 min, and blocked with 2% BSA for 30 min. Subsequently, the cells were incubated with the primary antibody at 4 °C overnight. Following this, the cells were treated with the secondary antibody for 1 h at room temperature, and the nuclei were stained with DAPI (Beyotime, China). Between each step, the cells were washed three times with TBST for 5 min each time. Images were captured using a FluoView 300 confocal laser scanning microscope (Olympus, Tokyo, Japan).
Western blot analysis of total cellular proteins
Proteins were separated by SDS-PAGE, followed by detection via Western blot. The specific methods are described in 2.10.
Statistical analysis
Statistical analyses were performed using SPSS 23.0 software. Multiple comparisons were conducted using one-way analysis of variance (ANOVA), followed by post-hoc analysis with Tukey’s test. Statistical significance was defined as p-values less than 0.05 within a 95% confidence interval. Results were visually represented using GraphPad Prism version 10.0.