Male BALB/c mice aged 6 weeks and weighing 18–20 g were purchased from the experimental animal center of Sun Yat-sen University. All animal experimental procedures were approved by the Sun Yat-sen University Committee for Animal Research and conformed to the Guide for the Care and Use of Laboratory Animals of the National Institute of Health in China.

Bone marrow-derived DCs were generated using methods described by Inaba et al. (1992) and Lutz et al. (1999). Briefly, BALB/c mice tibiae were removed and left in 70% ethanol for 2–5 min for disinfection and then washed in PBS. Cells within the marrow were disintegrated by vigorous pipetting. Thereafter, cells were cultured in medium RPMI-1640 (GIBCO, Germany) supplemented with Penicillin (100 U/ml, Sigma, Germany), Streptomycin (100 μg/ml, Sigma, Germany), L-glutamin (2 mM, Sigma, Germany), 10% heat-inactivated fetal calf serum (GIBCO, Germany), recombinant murine granulocyte–macrophage colony-stimulating factor (200 U/ml, perprotech, United States), recombinant murine interleukin-4 (5 ng/ml, perprotech, United States). At days 3, 5, 7 and 9, half of the cells culture supernatant was collected and centrifuged, the cell pellet resuspended and given back to the original plate, and then adding the equivalent of the medium. At day 10, cells can be used (BMDCs). The prepared BMDCs were treated with SEA (40 μg/ml) or untreated; after incubation for 24 h, the culture supernatant was harvested. Exosomes were purified from the supernatant using an exosome extraction kit (Invitrogen, United States) according to manufacturer’s instructions.

Negative-staining transmission electron microscopy (TEM) was conducted to analyze the exosomes. Exosomes suspended in 2% glutaraldehyde were loaded on a copper grid and negatively stained with 3% (w/v) aqueous phosphotungstic acid for 1 min. The grid was then examined using an FEI Tecnai G2 Sprit Twin transmission electron microscope.DC exosome particles were analyzed using NanoSight NS300 (Malvern Instruments, United Kingdom).

Dendritic cell exosomes were lysed in a western blotting lysis buffer, separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently electrotransferred onto a nitrocellulose blotting membrane (GE Healthcare Life Science, United Kingdom). The membranes were incubated with anti-mouse CD63, CD9, CD81 (BD Biosciences, United States) and Calnexin (Abcam, United Kingdom) antibodies and horseradish peroxidase (HRP) conjugated anti-mouse IgG (Jackson, United States), followed by chemiluminescent detection (Amersham, United States).

Acute colitis was induced via the administration of DSS (molecular mass 36–50 kDa; MP Biomedicals, Illkirch, France) in drinking water (5.0%, wt/vol). The control group received regular drinking water. Treatments with exosomes or SEA were conducted via intraperitoneal (i.p.) injection (50 μg per mouse) from day 0 to day 6. Control groups received the same volume of vehicle (PBS). There were five mice in each group, and each group was used for three independent experiments.

Mice were weighed daily, and the occurrence of diarrhea and bleeding were recorded. Scores were evaluated based on weight loss, stool type, and bleeding (Table 1) as described by Sann et al. (2013). Briefly, weight loss was scored as 0 (< 2%), 1 (≥ 2%– < 5%), 2 (≥ 5%– < 10%), 3 (≥ 10%– < 15%) or 4 (≥ 15%); stool was scored as 0 (normal), 1 (softer stool), 2 (moderate diarrhea), or 3 (diarrhea); and bleeding was scored as 0 (no rectal bleeding), 1 (weak hemoccult), 2 (blood in stool), or 3 (fresh rectal bleeding). The summed scores for weight loss, diarrhea, and bleeding were used as the clinical disease score [disease activity index (DAI)]. All scores were assessed by an independent observer blinded to the treatment.

On day 6, the animals were sacrificed, and their colons were removed. Colon length was measured as an indirect marker of inflammation. An independent observer who was blinded to treatment status determined the macroscopic characteristics. Briefly, the macroscopic scores were 0 (no damage), 1 (hyperemia without ulcers), 2 (hyperemia and wall thickening without ulcers), 3 (one ulceration site without wall thickening), 4 (two or more ulceration sites), 5 (0.5-cm extension of inflammation or major damage), and 6–10 (1-cm extension of inflammation or severe damage; Table 2; Yang et al., 2016).

Colons were fixed overnight in 4% (w/v) paraformaldehyde. Five-millimeter paraffin sections were stained with hematoxylin and eosin (H&E). The histopathological score was examined in a blinded fashion according to the criteria described by Sann et al. (2013). The following parameters were evaluated: extent of inflammation, infiltration of neutrophils and lympho-histiocytes, extent of crypt damage, crypt abscess, sub-mucosal edema, loss of goblet cells, and reactive epithelial hyperplasia (Table 3). The aggregated individual histopathological scores were calculated.

RNA was extracted was by Trizol reagent (Invitrogen, United States), according to the manufacturer’s instructions. Total RNA (1000 ng) was converted to cDNA by a Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, United States). PCR reactions were performed using SYBR Green QPCR Master Mix (TaKaRa, Japan), according to manufacturer’s instructions. The reaction over 40 cycles with denaturation at 95°C for 30 s followed by 95°C for 5 s and 60°C for 20 s. The primers of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-17 (IL-17A), interleukin-12 (IL-12), interleukin-22 (IL-22) and transforming growth factor (TGF)-β are listed in Table 4.

Data were expressed as means ± SEM. Multiple groups were compared using a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison post hoc test to determine statistical significance. Statistical significance between two experimental groups was assessed using the Independent-Sample t-test. A value of P < 0.05 was considered as significant.

Bone marrow-derived DCs were cultured in vitro and either treated with SEA or left untreated. Culture supernatants were collected after 24 h. Exosomes were isolated from supernatants using exosome extraction kits according to the manufacturer’s instructions. To verify the purity and quality of the exosomes, negative-staining TEM was conducted to evaluate exosome morphology. Negative-staining TEM revealed that most untreated DC exosomes and SEA-treated DC exosomes displayed closed round vesicles with diameters of 30–150 nm (Figure 1A), indicating that the exosomes were undamaged during purification and uncontaminated with microparticles of cellular organelles or membranes. Furthermore, to test the expression of the exosome-specific markers, CD63, CD9, and CD81 were confirmed via western blotting; these markers were expressed at high levels (Figure 1B). Calnexin, the negative marker of exosomes also be determined by western blotting to test the purity of exosomes (Figure 1B). In addition, the size distribution profile of the exosomes was investigated using NanoSight, which revealed a size peak of 108 nm among untreated DC exosomes and 104 nm among SEA-treated DC exosomes (Figure 1C). These results indicate the successful isolation of the exosomes from the culture supernatants.

To test whether SEA-treated DC exosomes attenuated colitis, acute DSS-induced colitis mice were injected i.p. with SEA-treated DC exosomes (50 μg/mouse) from day 0 to day 6. Clinical scoring was based on weight loss, diarrhea, and bleeding and expressed as the DAI. DSS treatment induced substantial weight loss in mice. After DSS administration, the mice of the DSS+PBS group lost weight over time, with a maximum body weight loss of 26.75% within 6 days (Figure 2A). Compared with the DSS+PBS group, weight loss in the colitis mice treated with SEA-treated DC exosomes was significantly alleviated (14.31% vs. 26.75% on day 6; P < 0.05). In addition, compared with untreated DC exosomes and SEA, SEA-treated DC exosomes better alleviated body weight loss (Table 5). Second, DSS treatment caused severe diarrhea and bleeding that persisted until the sacrifice of the colitis mice. Untreated DC exosomes, SEA-treated DC exosomes and SEA alleviated the severity of diarrhea and bleeding; however, the effect of SEA-treated DC exosomes was greater than that of DC exosomes and SEA. The DAI (the sum of the weight loss, diarrhea and bleeding scores) of the colitis mice treated with SEA-treated DC exosomes was lower than that of the colitis mice treated with PBS, SEA-untreated DC exosomes and SEA (Figure 2B and Table 5). These results suggest that SEA-treated DC exosomes attenuate clinical scores in DSS-induced colitis mice, and this effect was greater than that of SEA-untreated DC exosomes and SEA.

Dextran sulfate sodium significantly reduces colon length in colitis mice. Therefore, the therapeutic potential of SEA-treated DC exosomes in suppressing acute colitis was investigated using colon length. The results showed that compared with the colitis mice treated with PBS, the colons of the colitis mice treated with SEA-treated DC exosomes were longer (8.2 ± 1.3 cm vs. 5.5 ± 1 cm, P < 0.01; Figures 3A,B). In addition, the colons of colitis mice treated with SEA-treated DC exosomes were longer than those in mice treated with SEA-untreated DC exosomes and SEA (Figures 3A,B and Table 5). Mean colon macroscopic scores were assessed based on hyperemia, wall thickening, ulceration, inflammation extension, and damage. Compared with the DSS+PBS group, the mean colon macroscopic scores of colitis mice treated with SEA-treated DC exosomes were significantly suppressed (Figure 3C and Table 5). In addition, the mean colon macroscopic scores of colitis mice treated with SEA-treated DC exosomes were lower than those of colitis mice treated with SEA-untreated DC exosomes and SEA (Figure 3C and Table 5). These data demonstrate that SEA-treated DC exosomes ameliorate reduction in colon length and macroscopic scores in acute DSS-induced colitis mice more effectively than DC exosomes untreated with SEA and SEA.

Finally, damage was assessed via a histologic examination. Colonic sections revealed the complete disruption of the colonic architecture in colitis mice. Colitis mice treated with SEA-treated DC exosomes retained intact colonic architecture, including reduced numbers of mucosal erosions, smaller ulcerations, lower hyperplasia, and decreased inflammatory infiltration. The damage severity in mice treated with SEA-untreated DC exosomes and SEA were greater than that in mice treated with SEA-treated DC exosomes (Figure 4A). Consistent with the histologic examination, the histological scores of colitis mice treated with SEA-treated DC exosomes were significantly lower than PBS-treated mice, SEA-untreated DC exosomes treated mice and SEA-treated mice (Figure 4B and Table 5). Taken together, these results indicate that SEA-treated DC exosomes prevent colon damage in acute DSS-induced colitis mice, and this effect was greater than that achieved with SEA-untreated DC exosomes and SEA.

Inflammatory cytokines, such as TNF- α, IFN-γ, IL-17A, IL-12, IL-22 and TGF-β, play important roles in the pathogenesis of IBD. To determine whether SEA-treated DC exosomes can regulate the inflammatory cytokines production in colitis mice colon, the expression levels of inflammatory cytokines were examined by Real-time PCR. The results demonstrated that proinflammatory cytokines TNF- α, IFN-γ, IL-17A, IL-12, IL-22 were high expression levels in the DSS+PBS group (Figure 5). Compared with DSS+PBS group, TNF- α, IFN-γ, IL-17A, IL-12, IL-22 were reduced by treatment with SEA-treated DC exosomes (Figure 5). Furthermore, the expression levels of TNF- α, IFN-γ, IL-17A, IL-12, IL-22 in DSS+SEA-EXO group were lower than DSS+SEA group and DSS+EXO group (Figure 5). In addition, the expression levels of anti-inflammatory cytokines TGF-β in DSS+SEA-EXO group was higher than other groups (Figure 5). These results indicate that SEA-treated DC exosomes regulate inflammatory cytokines production in acute DSS-induced colitis mice.