Bone mesenchymal stem cell-derived exosomes prevent hyperoxia-induced apoptosis of primary type II alveolar epithelial cells in vitro

BMSC isolation and culture

Animal experiments were performed with approvals from the Laboratory Animal Welfare Ethics Committee of the Medicine Center at Shenzhen Peking University and Hong Kong University of Science and Technology, China (approval number: 2021-147). For the experiments, sixteen 6-week-old Sprague Dawley rats weighing about 200 g (Experimental Animal Center of Southern Medical University, Guangzhou, China) were housed in steel cages at 18–22 °C under 50–60% relative humidity. A standard pellet diet and water were provided ad libitum. The rats were sacrificed by cervical dislocation after 1 week, and the femur and tibia were collected under aseptic conditions. The bone marrow cavity was washed with Dulbecco’s modified Eagle medium (DMEM; Gibco, Lifetech, China), filtered and centrifuged at 1,000 rpm for 5 min. Next, the BMSCs were resuspended in DMEM containing 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), seeded (1 × 10⁶/mL) in 10-cm culture plates, and cultured for 3 days before refreshing half of the medium with fresh culture medium. At approximately 80–90% confluency (after ∼10 days of cell culture), the BMSCs were trypsinized (0.25% trypsin, Gibco, Thermo Fisher Scientific, Waltham, MA, USA). BMSCs from passages 4 to 8 were used for subsequent experiments. The osteogenic and adipogenic potentials of the stem cells were determined by Alizarin red staining and oil red staining, respectively, as described previously (Futrega et al., 2018). The same differentiation medium and staining solution were used for skin fibroblasts as the negative control group.

Isolation of AECIIs from pregnant rats and culture grouping

The AECIIs were isolated from six 4-week-old pregnant Sprague Dawley rats weighing approximately 80–100 g (Experimental Animal Center of Southern Medical University, Guangzhou, China). Primary AECIIs were isolated as previously described (Gonzalez & Dobbs, 2013), with modifications. Briefly, pregnant rats at 20 days of gestation were first anesthetized intraperitoneally with 3% pentobarbital sodium (50 mg/kg; Shanghai Sangong Bioengineering, Shanghai, China) and then subjected to uterine-incision delivery to induce premature labor. Fetal lungs were removed from the thoracic cavity, rinsed with precooled phosphate-buffered saline (PBS), diced into small pieces, and trypsinized(0.25% trypsin) for 25 min at 37 °C. DMEM containing 10% FBS was added to the cell suspension to stop the digestion, and then the cells were filtered and centrifuged at 1,000 rpm for 5 min. The cell pellets obtained were incubated with collagenase IV (10 µg/mL) for 15 min at 37 °C. DMEM containing 10% FBS was then added to stop the reaction. Next, the cell suspension was centrifuged, resuspended in DMEM, and then inoculated in a 10-cm culture plate. After the second passage, the primary AECIIs were incubated with the AECII-specific surface marker surfactant protein C (SP-C) antibody followed by the corresponding FITC-conjugated secondary antibody for detection by confocal microscopy (Leica). We also detected lamellar bodies in ACEIIs using a JEOL 1400 transmission electron microscope. The isolated AECIIs were divided into five culture conditions: normal control (NC; 21% oxygen), hyperoxia (85% oxygen), hyperoxia+exosome (20 µg/mL), hyperoxia+exosome+LY294002 (a PI3K/Akt inhibitor, Sigma, Shanghai, China; 20 µM), and hyperoxia+exosome+rapamycin (an mTOR inhibitor, Sigma, Shanghai, China; 5 nM).

Isolation and characterization of BMSC-derived exosomes

BMSC-derived exosomes cultured at 37 °C under 5% CO₂ for 12 h were harvested from FBS-free culture medium using Total Exosome Isolation Reagent (Life Technology, Shanghai, China), according to the manufacturer’s instructions. The isolated BMSC-derived exosomes were then washed three times with PBS. As described previously (Zhang & Chan, 2010), the BMSC-derived exosomes were deposited on Formvar/carbon-coated electron microscopy grids and allowed to air dry. Subsequently, the BMSC-derived exosomes were fixed with 2% glutaraldehyde, counterstained with 4% uranyl acetate, and then scanned using a JEOL 1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan). The BMSC-derived exosomes were assessed by detection of two well-characterized exosome surface protein markers, CD9 and CD63, through nanoparticle tracking analysis with a ZetaView^® Nanoparticle Tracking Analyzer (Particle Metrix, Meerbusch, Germany) and western blot analysis.

Cell counting kit-8 (CCK8) cell viability assay

AECIIs (5 × 10³ cells/well) were seeded in 96-well plates and cultured in DMEM containing 10% FBS for 6 h under 85% oxygen before exosomes and/or LY294002 or rapamycin inhibitor were added. After culturing for 24, 48, and 72 h, respectively, the AECII cell viability was assessed at 490 nm by the CCK8 assay (Beyotime Biotech, Shanghai, China), according to the manufacturer’s instructions. Briefly, AECII cultures were incubated with 0.5 mg/mL CCK8 for 20 min before measurements were taken.

Annexin V-FITC apoptosis assay

AECII apoptosis was assessed using an annexin V-FITC apoptosis detection kit (Beyotime Biotech, Jiangsu, China). After treatment with exosomes and/or LY294002 or rapamycin inhibitor for 24 h, AECIIs were spun down and incubated for 15 min at room temperature in the dark. Next, the cell pellets were resuspended in 500 µL of binding buffer containing 5 µL of annexin V-FITC and 5 µL of propidium iodide. The AECIIs were prepared using a flow cytometry assay kit (Abcam, Cambridge, UK) and were then counted using a BD Accuri C6 Plus flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Western blotting

Total protein was extracted from AECIIs treated with BMSC-derived exosomes and/or inhibitors using radioimmunoprecipitation assay lysis buffer (Beyotime Biotech, Shanghai, China). The protein concentration was determined using a bicinchoninic acid kit (Beyotime Biosciences, Shanghai, China). Proteins were separated by gel electrophoresis, and the bands were transferred onto polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membranes were first incubated with mouse anti-human AKT, p-AKT, p-mTOR, mTOR, Bax, Bcl-2, or Ki67 primary antibodies (Santa Cruz, USA) at 4 °C overnight, followed by the corresponding anti-rabbit and anti-mouse secondary antibodies (BOSTER Technology, Wuhan, China) at room temperature for 1 h. The bands were visualized using the enhanced chemiluminescence substrate and were then analyzed using ImageJ software (NIH, Bethesda, MD, USA). GAPDH (Sanjian Biology, Tianjin, China) was used as the loading control.

Statistical analysis

Statistical analysis was performed using SPSS v20 software. Data from three independent experiments were expressed as the mean  ± standard deviation (SD). Comparison among groups was performed by one-way analysis of variance, while the two-tailed Student’s t-test was used to compare groups. Values of P < 0.05 were considered statistically significant.

Morphology and stem cell characteristics of BMSCs

Unlike the BMSCs seeded at day1 (Fig. 1A), the cells appeared as fibroblast-like mononuclear cells and underwent logarithmic growth in the second passage (Fig. 1B). The adipogenic and osteogenic potential of the BMSCs were confirmed by oil red staining (Figs. 1C and 1D) and Alizarin red staining (Figs. 1E and 1F), respectively, indicating successful isolation of rat BMSCs. Fibroblasts were not positively stained by either oil red or Alizarin red (Figs. 1D and 1F).

Characterization of BMSC-derived exosomes

From transmission electron microscopy,we observed particles with diameters of 40–100 nm, which is characteristic of exosomes (Fig. 2A). Vesicles with an average particle diameter size of 100–200 nm and a maximum diameter of 600 nm were detected from nanoparticle tracking analysis (Fig. 2B). Western blot analysis also confirmed successful isolation of BMSC-derived exosomes (Fig. 2C).

Morphology and characterization of ACEIIs

Typical lamellar bodies were detected by transmission electron microscopy (Fig. 2D). AECIIs formed colonies and exhibited a cuboidal cobblestone morphology under a light microscope (Fig. 3A). Detection of the commonly expressed AECII marker SP-C by immunofluorescence also confirmed successful AECII isolation (Figs. 3B, 3C and 3D).

BMSC-derived exosomes protect AECIIs from hyperoxia-induced apoptosis

Next, we determined the effect of exosomes on AECII cell viability and apoptosis under hyperoxia. The CCK8 assay showed no significant changes in cell viability for all groups at 24 h, but the cell viability was significantly decreased in the hyperoxia group at 48 h (t = 6.3246, P = 0.0032) and 72 h (t = 16.5132, P = 0.0001) compared with the normal control group (Fig. 3E). Of note, hyperoxia-mediated growth inhibition was significantly reduced in the hyperoxia+exosome group at 48 h (t = 7.8558, P = 0.0014) and 72 h (t = 10.4151, P = 0.0005), compared with the hyperoxia group (Fig. 3E). The hyperoxia+exosome+LY294002 and hyperoxia+exosome+rapamycin groups showed significantly lower cell viabilities, compared with the hyperoxia+exosome group at 48 h (Fig. 3E; hyperoxia+exosome+LY294002 vs.hyperoxia+exosome, t = 8.8822, P = 0.0009; hyperoxia+exosome+rapamycin vs.hyperoxia+exosome, t = 9.8590, P = 0.0006) and 72 h (Fig. 3E, hyperoxia+exosome+LY294002 vs.hyperoxia+exosome, t = 11.4614, P = 0.0003; hyperoxia+exosome+rapamycin vs.hyperoxia+exosome, t = 9.3915, P = 0.0007).

There were significant differences of early and late apoptosis or necrosis of AECIIs between the normal control group (Fig. 4A) and the hyperoxia group (Fig. 4B, t = 27.3758, P = 0.0000). In addition, a marked decrease in apoptotic and necrotic AECIIs was observed for the hyperoxia+exosome group (Fig. 4C, t = 22.8889, P = 0.0000); however, the effects were reversed for the hyperoxia+exosome+LY294002 group (Fig. 4D, t = 15.6880, P = 0.0001) and the hyperoxia+exosome+rapamycin group (Fig. 4E, t = 27.0877, P = 0.0000).

Exosomes affect the levels of apoptosis-related proteins and the phosphorylation of key factors of the PI3K/Akt/mTOR signaling pathway

The effect of BMSC-derived exosomes on hyperoxia-induced apoptosis of AECIIs was assessed by western blot analysis (Figs. 5A and 5B). The Bax/Bcl-2level was significantly increased, while the Ki67 level was decreased in the hyperoxia group, compared with the normal control group (Figs. 5A and 5B; t = 6.1929, P = 0.0035; t = 3.3567, P = 0.0284, respectively). In addition, Akt, PI3K, and mTOR phosphorylation was inhibited in the hyperoxia group but was rescued in the hyperoxia+exosome group (Figs. 5A and 5B; t = 25.5119, P = 0.0000; t = 9.2418, P = 0.0008; t = 9.0355, P = 0.0008, respectively). Furthermore, decreased p-Akt and Ki67 protein levels as well as mTOR phosphorylation were noted in the hyperoxia+exosome+LY294002 group, compared with the hyperoxia+exosome group (Figs. 5A and 5C; t = 21.4586, P = 0.0000; t = 6.5485, P = 0.0028; t = 14.1400, P = 0.0001, respectively).