2.1. BMSC isolation, expansion, and identification
All animals used in this study were purchased from Hubei Bente Biotechnology Co., Ltd. (Wuhan, China). The entire research process was supported and supervised by the Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology, China. BMSCs were isolated from male Sprague Dawley rats weighing 60–100 g according to previously described methods [24, 25]. Briefly, the bone marrow in the rat femur and tibia flushed out using a 5-mL sterile syringe was cultured in DMEM/F12 (Gibco, Grand Island, NY, USA) containing 10% exosome-depleted fetal bovine serum (Gibco, Australia) in a humidified atmosphere of 5% CO2. After culturing the cells for 48 h, the medium was changed to remove non-adherent cells. When the cells reached 80–90% confluence, they were digested with trypsin (0.05% Trypsin-EDTA, Gibco, Canada) and then subcultured and expanded. At passages 3–5 (P3–P5), the cells were labeled with antibodies against CD29, CD90, CD45, and CD11b (BioLegend, San Diego, CA, USA), and BMSCs were phenotyped by flow cytometry.
2.2. Construction of recombinant lentivirus carrying EphB2
We used GenBank to design forward (5′-GTCAACACGCTGGACAAGAT-3′) and reverse (5′-CCTTATAGTCCTTATCATCGTC-3′) primers, which were synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China). After the amplified EphB2 gene fragment was inserted into the lentiviral vector GV342 (Ubi-MCS-SV40-puromycin), the linearized GV342 vector was used to amplify the EphB2 gene fragment using polymerase chain reaction (PCR). Positive clones with 394-bp PCR products were selected for sequencing. This recombinant lentivirus was designed with both the puromycin-resistant gc green fluorescent protein gene. After co-transfection with GV492 and packaging plasmids, pHelper 1.0 and pHelper 2.0 (Genechem), recombinant lentivirus was produced by 293T cells.
2.3. Lentiviral transfection of BMSCs
P2–P3 BMSCs were inoculated into the culture flask and cultured for approximately 8–12 h until reaching 30–40% confluence, following which transfection was performed at a multiplicity of infection of 30–35 using an appropriate dose of HitransG P, according to the manufacturer’s instructions. The genetically engineered BMSCs used recombinant lentiviruses that simultaneously expressed EphB2 and puromycin-resistant genes. The negative control used an empty lentivirus with anti-puromycin gene but not the EphB2 gene. Approximately 48–72 h after transfection, the medium containing lentivirus was discarded and replaced with complete medium. To screen for the stable expression of EphB2-BMSCs, puromycin was added to the complete medium to a final concentration of 2–3 mg/mL, and the medium was changed daily for 7 days. The obtained stably transfected cells were expanded to generations P4–P5 and used for exosome extraction.
2.4. Exosome isolation, purification, and identification
To prepare the exosomes, we cultured P4–P5 BMSCs to approximately 80–90% confluence and then cultured the cells in serum-free DMEM/F12 for 48 h to obtain more exosomes, following which the cell culture supernatants were collected. Exosomes were acquired by ultracentrifugation, first by centrifuging the cell culture supernatant at 300 ×g for 15 min and then at 2000 ×g for 30 min. The obtained supernatant was centrifuged at 12,000 ×g for 30 min. To remove excess cell debris, the supernatant was filtered through a 0.22-µm filter. The supernatants were ultra-centrifuged twice (Beckman, Brea, CA, USA) at 120,000 ×g for 90 min, which yielded the pure exosomes. All the above steps were conducted at 4°C (Fig. 1).
To demonstrate the successful isolation of exosomes, the morphology of the enriched exosomes was observed using transmission electron microscopy (TEM) (Tecnai, FEI, Hillsboro, OR, USA). The marker proteins on exosomes (CD9, CD63, and TSG101) were detected by western blotting. The size distribution of exosomes was measured using nanoparticle tracking analysis (NanoSight, Salisbury, UK). After quantifying the protein concentration of exosomes using a BCA kit (Beyotime Biotechnology, China), the exosomes were stored at -80°C until analysis.
2.5. Cell culture
Caco-2 cells were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co. (Shanghai, China) and cultured in high-glucose (HG)-DMEM (Gibco, Waltham, MA, USA) containing 10% FBS, 2.5% HEPES (Sigma, St. Louis, MO, USA), and 1% antibiotics (Sigma-Aldrich, St Louis, MO, USA) and seeded onto culture flasks (25 or 75 cm2). The complete medium was replaced every two days; when the cells were 90% confluent, they were digested with 0.25% trypsin-EDTA for cell subculture. This cell line is commonly used in studies of intestinal epithelial wound healing [26].
2.6. CCK-8 assay
The effect of EphB2-Exos on the viability of Caco-2 cells was evaluated in a Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) assay. After Caco-2 cells were seeded onto 96-well plates and cultured for 12 h, the complete medium was replaced with HG-DMEM containing 2% serum, and exosomes and EphB2-Exos were added to the medium. After 24 h of treatment, CCK-8 reagent with a final concentration of 10% was added to the 96-well plate. After incubation for 2 h at 37°C, the amount of formazan formed was detected by measuring the absorbance at 450 nm with a reference wavelength of 655 nm using a multimode plate reader (PerkinElmer EnSpire Plate Reader, Waltham, MA, USA).
2.7. Wound‑healing assay
Caco-2 cells were seeded onto a six-well plate at a density of 3 × 105 cells per well. After the cells reached around 90% confluence, scratch analysis was performed. The scratches in each well were created using a sterile 10-µL pipette tip. The scraped floating cells were washed twice with PBS, and the remaining cells were incubated with EphB2-Exos. An optical microscope (Olympus, Tokyo, Japan) was used to photograph the same locations along the scratch wound at 0 and 24 h. ImageJ software 8.0 (NIH, Bethesda, MD, USA) was used to measure the areas of the scratch wounds.
2.8. Induction of oxidative stress in Caco-2 cells
The culture medium of Caco-2 cells grown in six-well plates was collected to detect peroxides and antioxidant enzymes. The cells were pretreated with exosomes and EphB2-Exos for 12 h. To mimic oxidative damage, in addition to the control group, 250 µM hydrogen peroxide (H2O2, Sigma-Aldrich, St. Louis, MO) was added to the Caco-2 cells in each group. The experimental groups were as follows: (1) control, (2) H2O2 (250 µmol/L), (3) exosomes (30 µg/mL), and (4) EphB2-Exos (30 µg/mL). After treatment for 24 h, the cells were collected and used to detect the concentrations of myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), and nitric oxide (NO) using a commercial assay kit (Nanjing Jiancheng, China), and the cell culture media from each group were collected for subsequent experiments.
2.9. Measurement of intracellular reactive oxygen species levels
The reactive oxygen species (ROS) generation test was performed as previously described [27]. For the experiment, 3 × 105 Caco-2 cells per well were seeded into a 6-well black plate in complete culture medium. The groups of cells were prepared and treated as described previously. After these steps, the medium was aspirated, and cells were washed twice with PBS. Next, 1 mL of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was added to each well at a concentration of 10 µM, and the cells were further incubated at 37°C under 5% CO2 for 60 min. After incubation, DCFH-DA was removed, and the cells were washed with PBS. The negative controls contained cells in DMEM, whereas positive controls contained cells in DMEM with 20 µM hydrogen donor for 60 min. The medium was aspirated, and the cells were collected with PBS. These suspensions were centrifuged at 300 ×g for 5 min; the pellet was washed twice with PBS and then suspended in sterile PBS. The fluorescence of the cells was assessed by flow cytometry. The average 2′,7′-dichlorofluorescein (DCF) fluorescence was determined as a percentage compared to the negative control, which was assumed to be 100%.
2.10. Enzyme-linked immunosorbent assay
Cell culture medium samples were obtained from the different groups, and the protein levels of IL-1β, IL-6, IL-17A, IL-10, and TGF-β1 in the media were measured using enzyme-linked immunosorbent assay kits (Elabscience Biotechnology Co., Ltd., Houston, TX, USA) according to the manufacturer’s instructions.
2.11. Fluorescein isothiocyanate-dextran permeability assay
To measure intestinal barrier function, we seeded 5 × 104 Caco-2 cells onto the apical side of 24-well Transwell polyester membrane filters (6.5 mm diameter, 3 µm pore size; Corning, Inc., Corning, NY, USA) and cultured the cells for at least 21 days. Cells in the upper chamber were pretreated with exosomes or EphB2-Exos for 24 h, and 3% DSS dissolved in DMEM was added to the upper chamber for 8 h. To determine the permeability of cell monolayers, the cells were incubated with fluorescein isothiocyanate-dextran (FD-4, 4 kDa, 1 mg/mL; Sigma-Aldrich, St. Louis, MO) in the upper chamber for 2 h at 37°C; subsequently, 100 µL of samples from the outer chamber was collected for assessment using a multimode plate reader at excitation and emission wavelengths of 485 and 535 nm, respectively. The dextran concentration in each sample was calculated using a fluorescein isothiocyanate-dextran standard curve.
2.12. Western blotting
Caco-2 cell proteins were extracted using RIPA lysis solution containing a cocktail of protease inhibitors (Servicebio Technology, Wuhan, China). After treatment, the Caco-2 cells were washed twice with cold PBS, and RIPA lysis solution was added to the cells. The protein concentration was determined using a BCA protein assay kit (Beyotime Biotechnology, China). The same mass of protein was separated using 8–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies against EphB2 (1:500; Abcam, Cambridge, UK), EphrinB1 (1:500; Cell Signaling Technology Inc., Danvers, MA, USA), RhoA (1:2000; Abcam), ROCK1 (1:2000; Abcam), ROCK2 (1:2000; Abcam), occludin (1:1000; Abcam), ZO-1 (1:2000; Abcam) and β-actin (1:10,000; CST) overnight at 4°C. Next, the membranes were washed three times with PBS containing Tween 20 and incubated with the appropriate secondary antibody at 25°C for 1 h. Finally, the membranes were visualized according to the manufacturer’s instructions.
2.13. Isolation and culture of CD4 + T lymphocytes
CD4+T lymphocytes were isolated from the fresh spleens of male rats weighing 80 g using a CD4+ negative isolation kit (Stem Cell Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions. The cells were cultured in 96-well flat bottom plates (2 × 105 cells/well) in RPMI-1640 (Gibco, Eggenstein, Germany) containing 10% fetal bovine serum, 1% antibiotics (penicillin –streptomycin), 1% sodium pyruvate, and 1% Glutamax (Invitrogen, Burlington, ON, Canada).
2.13. Co-culture of EphB2-Exos and CD4 + T lymphocytes
The effect of EphB2-Exos on CD4+ T lymphocytes in vitro was investigated by co-culturing CD4+ T lymphocytes and exosomes. A 96-well flat-bottomed plate was pre-coated with anti-CD3 antibody (5 µg/mL, BD Biosciences, Franklin Lakes, NJ, USA) and anti-CD28 antibody (10 µg/mL, BD Biosciences) overnight at 4°C to activate the CD4+ T lymphocytes. Subsequently, CD4+ T lymphocytes were added to the plate, which was washed twice with PBS. After 4 h, exosomes or EphB2-Exos were added, whereas CD4+ T lymphocytes were untreated (negative controls). The cells were maintained at 37°C for 3 days in a humidified incubator with 5% CO2. After 72 h of incubation, the CD4+ T lymphocytes were stained with PE-Cy7-anti-CD4 antibody (BD Biosciences), eFluor450-anti-IL-17A antibody (eBioscience, San Diego, CA, USA), PE-anti-Foxp3 antibody (eBioscience), or APC-anti-IFN-γ antibody (BD Biosciences). The stained cells were analyzed using flow cytometry.
2.14. Statistical analyses
All experimental data are presented as the mean ± standard error of the mean. Comparisons between multiple groups were evaluated using one-way analysis of variance followed by Tukey’s post hoc test. Statistical analysis was performed using GraphPad Prism (GraphPad Prism 8.4.2, USA). P < 0.05 was considered statistically significant.