Human umbilical cord mesenchymal stem cell-derived exosomes alleviate experimental colitis by maintaining the intestinal barrier and remodeling macrophage polarization

Abstract

BACKGROUND

Inflammatory bowel disease (IBD) is a chronic, nonspecific inflammatory disorder of the gastrointestinal tract for which there are currently no safe and effective therapeutic options. Human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex) have emerged as a new treatment strategy for IBD owing to their tissue repair and immunoregulatory functions. However, the mechanisms by which hucMSC-Ex exerts their effects on IBD are not yet fully understood.

AIM

To investigate the therapeutic effects of intraperitoneal injection of hucMSC-Ex on intestinal inflammation, intestinal barrier function, and macrophage phenotype in a mouse model of IBD induced with dextran sulfate sodium (DSS).

METHODS

A mouse colitis model was constructed using 2.5% DSS. Three groups were established: Normal control group (NC), DSS model group (DSS), and DSS + hucMSC-Ex group (hucMSC-Ex) (n = 8/group). General indicators such as body weight change, disease activity index score, colon length, and spleen index were measured. Histopathological damage was evaluated by hematoxylin and eosin staining. The expression of inflammatory factors was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot, and enzyme-linked immunosorbent assay. Antioxidant stress indicators, including glutathione, superoxide dismutase, and the Nrf2/HO-1/GPX4 axis, were assessed using commercial kits, RT-qPCR, and Western blot. The intestinal mucus barrier and mechanical barrier were examined by immunofluorescence, RT-qPCR, and Western blot. Macrophage polarization was detected by immunofluorescence, flow cytometry, and RT-qPCR. The RAW264.7 cell line was used in vitro to verify the above findings. A signal transducer and activator of transcription (STAT) inhibitor was employed to demonstrate that hucMSC-Ex functions via the Janus kinase (JAK) 1/STAT1 signaling pathway.

RESULTS

We found that hucMSC-Ex ameliorated the clinical symptoms of DSS-induced colitis, which mimics human IBD, by reducing colonic inflammation, colonic goblet cell loss, and intestinal mucosal permeability, while promoting the transformation of immature pro-inflammatory macrophages into mature anti-inflammatory macrophages. In vitro, hucMSC-Ex also modulated phenotypic changes in macrophages after RAW264.7 cells were induced toward M1 or M2 phenotypes. Furthermore, hucMSC-Ex altered macrophage phenotypic transformation, thereby ameliorating experimental colitis through modulation of the JAK/STAT signaling pathway both in vivo and in vitro.

CONCLUSION

HucMSC-Ex partially alleviates experimental colitis by restoring the intestinal barrier and intestinal immune homeostasis. This finding provides new insights into the therapy of IBD.

Key Words: Inflammatory bowel disease; Human umbilical cord mesenchymal stem cells derived-exosomes; Intestinal barrier; Macrophage phenotypic transformation; Janus kinase/signal transducer and activator of transcription signaling

Core Tip: In this study, we found that human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex) ameliorated dextran sulfate sodium-induced colitis by reducing colonic inflammation and maintaining intestinal mucosa permeability, while promoting the transformation of immature pro-inflammatory macrophages into mature anti-inflammatory macrophages. In vitro, hucMSC-Ex also modulated the phenotypic changes in RAW264.7 cells. Furthermore, hucMSC-Ex altered the phenotypic transformation of macrophages, thereby ameliorating experimental colitis through modulation of the Janus kinase/signal transducer and activator of transcription signaling pathway in vivo and in vitro. This study not only provides guidance for further research on molecular mechanisms of inflammatory bowel disease (IBD), but also offers a theoretical basis for targeted therapy of IBD.

INTRODUCTION

Inflammatory bowel disease (IBD) is a chronic, non-specific inflammatory disorder of the gastrointestinal tract, encompassing two subtypes: Ulcerative colitis (UC) and Crohn’s disease. The multifactorial pathogenesis of IBD is widely acknowledged to involve a complex interplay of environmental factors and genetic predisposition, leading to dysregulated intestinal immune responses. This process is further influenced by the gut microbiota, which can trigger a cascade of clinical manifestations[1-3]. IBD was initially reported in Western countries; subsequently, with the progress of industrialization and urbanization, its incidence and prevalence have been increasing year by year in newly industrialized countries across Asia, the Middle East, and South America. IBD has evolved into a global disease, imposing a substantial economic burden and seriously affecting patients’ quality of life and well-being[4,5]. Currently, various therapeutic regimens are available for IBD, including 5-aminosalicylic acid, immunomodulators, thiopurines, monoclonal antibodies, and small-molecule chemical agents[6,7]. However, a substantial proportion of patients find these regimens ineffective or intolerable due to adverse reactions, and the efficacy and safety of existing drugs also present significant challenges for clinical research. Moreover, the clinical heterogeneity between UC and Crohn’s disease makes it difficult to identify an optimal treatment applicable to all patients[8,9]. Therefore, there is an urgent need to explore alternative therapeutic strategies capable of adapting to the complex intestinal milieu and its microbial environment[10].

Previous studies have shown that macrophages are the primary cells responsible for inducing colonic inflammation[11,12]. Upon activation by pro-inflammatory stimuli, monocytes migrate to inflamed sites and differentiate into macrophages, which exhibit varying M1-like or M2-like characteristics under the influence of chemokines and inflammatory factors[13,14]. M1-like macrophages produce pro-inflammatory cytokines and T helper (Th) 1 chemokines, participating in antigen presentation, T-cell activation, and the initiation of adaptive immune responses. M2-like macrophages release suppressive cytokines to dampen immune responses and alleviate inflammation[15,16]. The signal transducer and activator of transcription (STAT) signaling cascades are critical regulators of macrophage differentiation and function. For instance, phosphorylated STAT1 translocates to the nucleus and mediates M1-like macrophage polarization, whereas STAT3 drives M2-like macrophage polarization. Previous studies have demonstrated that dysfunction of the Janus kinase (JAK)-STAT signaling pathway, as well as genetic variations within this pathway, is associated with IBD. The dysregulation of macrophage polarization, particularly the shift toward a pro-inflammatory M1 phenotype, disrupts immune homeostasis within the intestinal mucosa. This imbalance serves as a key mediator of intestinal inflammation and plays a significant role in the progression of IBD. Numerous studies indicate that modulating macrophage polarization and the balance between M1-like and M2-like macrophages is crucial for immunotherapeutic strategies against IBD[16-19]. For example, molecules such as Tollip have been shown to modulate macrophage polarization, thereby potentially mitigating the inflammatory response and offering a therapeutic avenue for IBD[17,20].

Mesenchymal stem cells (MSCs), which can be derived from multiple tissues including adipose tissue, bone marrow, dental pulp, and umbilical cord, represent a novel treatment for IBD owing to their high self-renewal capacity, multi-directional differentiation potential, and ability to regulate immune homeostasis and microbial balance[21-25]. The characteristics of easy extraction and expansion, non-invasive collection procedures, low cost, abundant cell content, higher proliferation capacity, low risk of infection, and low immunogenicity make human umbilical cord MSCs (hucMSCs) more suitable for the treatment of IBD compared with MSCs from other sources[26,27]. However, despite the promising potential of hucMSC therapy, certain limitations remain, including limited engraftment and low survival rates, as well as risks of differentiation into unwanted cell lineages, ectopic tissue development, tumorigenicity, genetic alterations, and ethical and safety challenges[28]. Notably, evidence suggests that hucMSCs primarily exert their therapeutic effects through paracrine mechanisms mediated by exosomes[27,29]. HucMSC-derived exosomes (hucMSC-Ex) exhibit all the advantages of hucMSCs without their drawbacks and are easier to maintain in terms of biological activity during storage and transportation[30]. Therefore, hucMSC-Ex has received widespread attention for its potential to reduce inflammation, promote tissue recovery, exert immunoregulatory effects, and cross biological barriers. Studies have indicated that hucMSC-Ex can effectively alleviate IBD through various regulatory mechanisms[31-33]. For instance, hucMSC-Ex inhibits lipid peroxidation and ferroptosis by targeting ACSL4 via miR-129-5p, while reducing intestinal inflammation and promoting tissue repair, thereby alleviating IBD[34]. HucMSC-Ex mitigates colitis induced with dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzenesulfonic acid by upregulating the expression of tumor necrosis factor-α-stimulated gene 6 (TSG-6), which restores the intestinal mucosal barrier and regulates the Th2/Th17 balance in mesenteric lymph nodes in mice[35]. HucMSC-Ex enriched with miR-378a-5p has been shown to alleviate DSS-induced colitis through inhibition of the NLRP3 inflammasome and suppression of macrophage pyroptosis[36]. However, given the role of macrophages in the progression of IBD, it remains to be explored whether and how hucMSC-Ex exert anti-inflammatory effects by reshaping intestinal macrophage development. In this study, we found that hucMSC-Ex can ameliorate DSS-induced colitis, promote intestinal barrier repair, enhance antioxidant capacity in macrophages, and modulate the phenotypic transition of macrophages. Further mechanistic studies indicated that hucMSC-Ex remodels macrophage phenotypic transition by regulating the JAK/STAT signaling pathway. These findings provide new insights into the repair mechanisms of hucMSC-Ex and contribute to a better understanding of their potential application in the clinical treatment of IBD.

MATERIALS AND METHODS

Exosome isolation and identification

HucMSCs were cultured in T125 flasks (NEST, China) until they reached 80% confluence. The supernatant was then removed, and the cells were gently washed twice with Dulbecco’s phosphate-buffered saline (PBS). Subsequently, the cells were cultured in serum-free medium for 24 hours to collect the supernatant. First, the supernatant was centrifuged at 300 g for 5 minutes, and then centrifuged at 2000 g for 20 minutes to remove cell debris. Second, the supernatant was centrifuged at 10000 g for 50 minutes to remove organelles and microvesicles for further purification of exosomes, followed by filtration through a 0.22 μm membrane filter. Next, the filtered supernatant was transferred to ultracentrifuge tubes and centrifuged at 100000 g at 4 °C for 2 hours. Finally, the supernatant was removed, and the resulting pellet was resuspended in PBS to obtain the exosomes. The prepared exosome solution could be used immediately or stored at -80 °C for future use. The extracted exosomes were characterized by transmission electron microscopy (TEM) to observe their morphology. Nanoparticle tracking analysis (NTA) was conducted to analyze particle size and capture video images of the exosomes. Expression of specific exosomal markers, including cluster of differentiation (CD) 81, caveolin-1, TSG101, and histone H3, was verified by Western blotting.

DSS-induced colitis

Male specific-pathogen-free C57BL/6J mice (6-8 weeks old, 21 ± 2 g) were purchased from the Experimental Animal Center of the Fourth Military Medical University (Xi’an, Shaanxi Province, China). They were acclimated for 7 days with tap water and a pelleted basal diet before the start of the experiments. The temperature was maintained at 23 ± 2 °C, with humidity at 50%–60% and a 12-hour light/dark cycle. Throughout the experiments, mice were fed a standard chow diet and tap water. The animal room was cleaned regularly during the housing period.

Mice were randomly allocated into three groups (n = 8/group): Negative control group (NC), DSS-induced colitis group (DSS), and hucMSC-Ex-treated colitis group (hucMSC-Ex). Mice in the NC group were fed tap water during the experiment, while mice in the DSS and hucMSC-Ex groups were fed tap water containing 2.5% DSS (molecular weight 36000-50000; MP Biomedicals, United States) for 7 consecutive days. A total of 200 μg of hucMSC-Ex was administered by intraperitoneal injection to mice in the hucMSC-Ex group on days 3, 6, and 9 of modeling, while mice in the other groups were injected with PBS. All mice were anesthetized with 5% isoflurane inhalation and then sacrificed by cervical dislocation on day 10. Disease progression in the different groups was evaluated by body weight change, disease activity index (DAI) score, and colon length. The gross appearance of the colon and spleen was observed and photographed, and the tissues were immersed in PBS pre-cooled to 4 °C. Colon, liver, and spleen tissues were fixed in 4% paraformaldehyde for preparation of paraffin sections, and the remaining colon tissues were used for follow-up experiments. All experimental procedures were approved by the Ethical Committee of the Fourth Military Medical University (approval No. IACUC-20240522).

Histological analysis

Sections of colon, liver, and spleen tissues were promptly fixed in 4% formalin overnight at room temperature, embedded in paraffin wax, stained with hematoxylin and eosin (HE), and subsequently scanned and analyzed with a pathological biopsy scanner. The histological score of the colon was determined as previously described[37].

Intestinal permeability assay

Serum was obtained by centrifuging blood for 10 minutes at 3500 rpm at 4 °C. Intestinal permeability was assessed by quantifying serum levels of intestinal fatty acid-binding protein (iFABP) according to the manufacturer’s detailed instructions using the iFABP enzyme-linked immunosorbent assay kit (EK1910, FineTest, Wuhan, Hubei Province, China).

Routine blood and biochemical parameters

Routine blood parameters, including white blood cell (WBC) count, red blood cell (RBC) count, and other relevant parameters, were measured using an automated 3-part differential hematology analyzer (Sysmex, Kobe, Japan). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined using an automated biochemical analyzer (Chemray 800, Shenzhen, Guangdong Province, China).

Myeloperoxidase activity assay

Neutrophil infiltration into colonic tissue was quantified by myeloperoxidase (MPO) activity measurement using an MPO assay kit (A044, Nanjing Jiancheng, Nanjing, Jiangsu Province, China) according to the manufacturer’s instructions. MPO activity was expressed as units per gram of total protein (U/g).

Determination of reduced glutathione and superoxide dismutase

Colonic tissues from each group were harvested and rinsed twice with pre-cooled PBS buffer, and the tissues were accurately weighed for subsequent use. Colon tissue homogenates were prepared according to the instructions of the reduced glutathione (GSH) kit (A006-2-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China) and the superoxide dismutase (SOD) kit (A003-1-2, Nanjing Jiancheng, Nanjing, Jiangsu Province, China).

Cell culture and hucMSC-Ex treatment

The mouse monocytic cell line RAW264.7 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell lines were tested for mycoplasma contamination before use in our experiments. RAW264.7 monocytes were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in 5% carbon dioxide. RAW264.7 cells were stimulated with mouse interferon (IFN)-γ (20 ng/mL, SinoBiology, China) and lipopolysaccharide (LPS) (100 ng/mL, Sigma, St. Louis, MO, United States) for 24 hours to induce the M1 phenotype (M^LPS), or with mouse interleukin (IL)-4 (20 ng/mL, SinoBiology, China) and IL-13 (20 ng/mL, SinoBiology, China) for 24 hours to induce the M2 phenotype (M^IL-4). Untreated RAW264.7 cells were used as the M0 phenotype. For treatment with hucMSC-Ex at a concentration of 4 μg/well, RAW264.7 cells were incubated for 24 hours after being treated with PBS, LPS + IFN-γ, or IL-4 + IL-13. After 24 hours of co-cultivation, cells and supernatants were collected for subsequent analysis. A phosphorylated STAT1 inhibitor (MedChem Express Co.) was used together with hucMSC-Ex after RAW264.7 cells were treated with different inducers in some experiments.

Western blot analysis

Radioimmunoprecipitation assay lysis buffer (Millipore, MA, United States) containing protease inhibitor and phosphatase inhibitor (Millipore, MA, United States) was used to extract total protein from colon tissue, RAW264.7 cells, and exosomes. Protein concentrations were quantified using the bicinchoninic acid protein assay kit (Yeasen, Shanghai, China). Equal aliquots of protein (40 μg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, United States). The PVDF membranes were blocked with 5% skimmed milk and then incubated overnight at 4 °C with a 1:1000 dilution of primary antibodies against claudin-1, occludin, zonula occludens-1 (ZO-1), E-cadherin, heme oxygenase-1 (HO-1), glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2 related factor 2 (Nrf2), JAK1, STAT1, phospho-STAT1 (p-STAT1), and β-actin antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. Protein signals were detected and visualized using a Bio-Rad Imaging System (ChemiDoc^TM, Bio-Rad, CA, United States).

Reverse transcription quantitative polymerase chain reaction

Total RNA from colon tissues and cells was isolated using TRIzol reagent (Invitrogen, United States) and chloroform extraction, and then reverse transcribed to complementary DNA using a PrimeScript RT Master Kit (TaKaRa, Shiga, Japan). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to measure transcript abundance of the target genes with SYBR Green Detection Mix (TaKaRa, Shiga, Japan) on a CFX Connect Real-Time PCR Detection System (Bio-Rad). Messenger RNA (mRNA) expression was normalized to the internal control β-actin. The RT-qPCR primers are shown in Supplementary Table 1.

Isolation of lamina propria cells and flow cytometry analysis

Lamina propria (LP) cells were isolated from colon biopsies according to the protocol. Briefly, colon biopsies were longitudinally cut and washed to remove feces, and then divided into 1 cm pieces. The biopsies were incubated in predigestion solution containing 5 mmol/L ethylenediaminetetraacetic acid and 0.145 mg/mL DL-dithiothreitol on a shaking platform for 20 minutes at 37 °C. The biopsies were then rinsed three times with PBS and passed through a 100 μm cell filter to remove epithelial cells. The remaining colon pieces were incubated for 25 minutes at 37 °C on a shaking platform in digestion medium containing 0.05 g collagenase D (Roche, Mannheim, Germany), 0.3 g dispase II (Roche, Mannheim, Germany), and 0.05 g DNase I (Sigma-Aldrich, Milan, Italy). After incubation, LP cells were enriched by Percoll density gradient centrifugation. The resulting cells were subsequently used for flow cytometry.

LP cells were stained with anti-CD45, anti-CD11b, anti-F4/80, anti-major histocompatibility complex II (MHCII), or anti-lymphocyte antigen 6C (Ly6C) for 30 minutes at 4 °C in the dark, and washed twice with washing buffer. The cells were then resuspended in 500 μL PBS and sorted on a fluorescence-activated cell sorter (FACS) Aria III flow cytometer (BD Immunocytometry Systems), and analyzed using FlowJo software (Ashland, OR, United States).

Immunostaining and confocal microscopy

For immunofluorescence (IF) staining, sections were stained with a mixture of primary antibodies against mucin 2 (Muc2), F4/80, CD206, and CD86. The slides were counterstained with 4’,6-diamidino-2-phenylindole (Vector Laboratories, Inc.) to identify nuclei. Images were acquired using an FV3000 laser scanning confocal microscope (Olympus, Inc.). The positive area was analyzed using CaseViewer.

Statistical analysis

All results are presented as the mean ± SD. Statistical analyses were performed using Student’s t-test for comparisons between two conditions or analysis of variance for comparisons among more than two groups, followed by post-hoc multiple comparisons (Dunnett’s test). Data analyses were performed using GraphPad Prism 8.0 software (GraphPad, San Diego, United States). P < 0.05 was considered statistically significant.

RESULTS

Isolation and characterization of hucMSC-Ex

The hucMSC culture supernatant was collected, and exosomes were extracted by ultracentrifugation according to a previously established method[38]. The purity and identity of hucMSC-Ex were assessed using NTA, TEM, and Western blot analysis. The NTA results revealed that hucMSC-Ex had an average diameter of approximately 135 nm (Figure 1A). TEM imaging indicated that hucMSC-Ex possessed an intact cell membrane, with low-density electron components within the vesicles and well-defined vesicular structures (Figure 1B). Western blot analysis confirmed the expression of surface marker proteins on hucMSC-Ex, including CD81, caveolin-1, and TSG101, whereas the negative marker histone H3 was not detected (Figure 1C). These findings demonstrated that hucMSC-Ex were successfully extracted, providing a material basis for subsequent experiments.

Figure 1 Identification of human umbilical cord mesenchymal stem cell-derived exosomes. A: Size distribution and particle concentration of hucMSC-Ex determined by nanoparticle tracking analysis; B: Morphological identification of hucMSC-Ex by transmission electron microscopy (scale bar = 200 nm); C: Western blot analysis of hucMSC-Ex surface markers. CD: Cluster of differentiation; hucMSC: Human umbilical cord mesenchymal stem cell; hucMSC-Ex: Human umbilical cord mesenchymal stem cell-derived exosomes.

HucMSC-Ex attenuates DSS-induced colitis in mice

To verify the reparative effect of hucMSC-Ex on DSS-induced colitis in mice, a colitis model was established using 2.5% DSS, and hucMSC-Ex were administered via intraperitoneal injection. The results indicated that mice in the NC group exhibited steady daily weight gain, whereas the DSS group began to experience sharp weight loss on day 6. In contrast, the hucMSC-Ex group showed moderate weight loss compared with the DSS group (Figure 2A). The DAI score, calculated based on body weight loss, fecal consistency, and fecal occult blood, serves as an indicator of intestinal inflammation severity. In the DSS-induced colitis model, the DAI score escalated rapidly from day 4, peaked on day 9, and subsequently decreased. In the hucMSC-Ex group, the increase in DAI score was more moderate throughout the entire experiment compared with the DSS group (Figure 2B). Gross observation and statistical analysis showed that colon length was shorter in the DSS group, whereas it was significantly restored in the hucMSC-Ex group (Figure 2C and D). Moreover, the spleen was enlarged in the DSS group, while splenomegaly was reduced in the hucMSC-Ex group (Figure 2E). The spleen index, used to evaluate systemic inflammation, was significantly higher in the DSS group than in the hucMSC-Ex group (Figure 2F). HE staining of colon tissues revealed that hucMSC-Ex treatment significantly alleviated the inflammatory response compared with the DSS group (Figure 2G). Blinded histological scores of the distal colon were significantly decreased in the hucMSC-Ex group relative to the DSS group (Figure 2H). Furthermore, we measured MPO activity, an index of neutrophil infiltration, and found a significant reduction in the hucMSC-Ex group compared with the DSS group (Figure 2I). Previous studies have shown that pro-inflammatory cytokines are elevated in DSS-induced colitis. Therefore, we further examined pro-inflammatory factors in colon tissues by RT-qPCR. The results showed that the mRNA expression levels of IL-12 and IL-18 were markedly increased in the DSS group, whereas hucMSC-Ex substantially decreased their expression (Figure 2J and K). Meanwhile, hucMSC-Ex dramatically increased the mRNA expression of the anti-inflammatory cytokines TGF-β and IL-4 (Figure 2L and M). These observations indicated that hucMSC-Ex mitigates the symptoms and tissue damage in DSS-induced colitis in vivo.

Figure 2 Human umbilical cord mesenchymal stem cell-derived exosomes attenuate dextran sulfate sodium-induced colitis in mice. A: Daily body weight changes in each group (dextran sulfate sodium vs human umbilical cord mesenchymal stem cell-derived exosomes); B: Disease activity index (DAI) scores in each group; C: Gross appearance of representative colons from mice in each group; D: Colon lengths of mice in each group; E: Macroscopic appearance of spleens from mice in each group; F: Spleen index of mice in each group; G: Representative histopathological changes in colon tissues assessed by hematoxylin and eosin staining (20 ×, scale bar = 200 μm; 40 ×, scale bar = 100 μm); H: Histological score of colons in each group; I: Colonic myeloperoxidase activity in each group; J-M: Messenger RNA expression levels of the inflammatory cytokines IL-12, IL-18, TGF-β, and IL-4. Data are presented as the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. Each experiment was repeated three times independently (n = 8 mice per group). NC: Negative control; DSS: Dextran sulfate sodium; MPO: Myeloperoxidase; IL: Interleukin; mRNA: Messenger RNA; TGF: Transforming growth factor; hucMSC-Ex: Human umbilical cord mesenchymal stem cell-derived exosomes.

HucMSC-Ex alleviates intestinal mucosal barrier dysfunction in DSS-induced colitis mice

Serum iFABP concentration was quantified to evaluate intestinal permeability after DSS administration. iFABP is located within the epithelial cells of the colon and is released into the bloodstream when mucosal permeability increases, serving as a sensitive indicator of intestinal barrier dysfunction. The results demonstrated that hucMSC-Ex administration significantly reduced intestinal permeability compared with the DSS group (Figure 3A). Elevated intestinal permeability suggests damage to the intestinal mucosal barrier. The protective effect of hucMSC-Ex on the intestinal mucosal barrier was evaluated by detecting the expression of tight junction (TJ) proteins, adhesion junction proteins, and Muc2 using RT-qPCR, Western blot, and IF. RT-qPCR, Western blot, and quantitative analysis of Western blot results showed significantly higher mRNA and protein expression of the TJ proteins ZO-1, claudin-1, and occludin, as well as the adhesion junction protein E-cadherin, in the hucMSC-Ex group compared with the DSS group (Figure 3B-J). Moreover, the mRNA levels of Muc2 were significantly increased in the hucMSC-Ex group relative to the DSS group (Figure 3K). Additionally, IF analysis revealed a significant increase in Muc2 staining in the hucMSC-Ex group compared with the DSS group (Figure 3L and M). These results showed that hucMSC-Ex exerts a protective effect on the intestinal mucosal barrier.

Figure 3 Human umbilical cord mesenchymal stem cell-derived exosomes alleviate intestinal mucosal barrier dysfunction in dextran sulfate sodium-induced colitis mice. A: Serum levels of intestinal fatty acidbinding protein (iFABP) as an indicator of intestinal mucosal permeability; B-E: Messenger RNA expression levels of the tight junction proteins ZO-1, claudin-1, and occludin, and the adhesion junction protein Ecadherin; F: Representative Western blot bands showing protein expression of ZO-1, claudin-1, occludin, and E-cadherin; G-J: Quantitative analysis of protein expression for ZO-1, occludin, claudin-1, and E-cadherin by Western blot; K: Messenger RNA expression level of mucin 2 (Muc2) (20 ×, scale bar = 200 μm; 40 ×, scale bar = 100 μm); L and M: Immunofluorescence staining for Muc2 in colonic sections. Data are presented as the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. Each experiment was repeated three times independently (n = 8 mice per group). NC: Negative control; DSS: Dextran sulfate sodium; hucMSC-Ex: Human umbilical cord mesenchymal stem cell-derived exosomes; NS: No significance; mRNA: Messenger RNA; ZO-1: Zonula occludens-1; iFABP: Intestinal fatty acid-binding protein; DAPI: 4’,6-diamidino-2-phenylindole; Muc2: Mucin 2.

HucMSC-Ex upregulates the Nrf2/HO-1/GPX4 axis to reduce inflammation and oxidative stress

We evaluated the overall inflammatory status of the mice through blood routine, biochemical indicators, and HE staining of the liver and spleen (Figure 4). The results indicated that WBC and platelet counts were elevated in the DSS group but decreased in the hucMSC-Ex group (Figure 4A and B). Compared with the DSS group, RBC counts were higher in the hucMSC-Ex group (Figure 4C). HE staining of the liver and spleen revealed that tissue structure was significantly disorganized in the DSS group, whereas the hucMSC-Ex group exhibited clearer and more intact tissue architecture. The margins of splenic nodules were blurred with indistinct boundaries in the DSS group, but were largely restored in the hucMSC-Ex group. In addition to elevated ALT and AST levels, pathological changes including hepatocyte swelling, eosinophilic degeneration, and inflammatory cell infiltration in the liver were observed in the DSS group; however, hucMSC-Ex significantly reduced these indicators (Figure 4D-F). Previous studies have shown that UC development is associated with increased inflammation and oxidative stress. Nrf2 is a transcription factor that responds to oxidative stress and plays a key role in regulating antioxidant processes. Activation of Nrf2 and subsequent upregulation of HO-1 and GPX4 can reduce oxidative stress. We measured the expression of the antioxidant substances GSH and SOD in colon tissues. The results showed that, compared with the DSS group, the hucMSC-Ex group significantly upregulated GSH and SOD expression (Figure 4G and H). Subsequently, we performed Western blot and RT-qPCR to examine the expression of Nrf2/HO-1/GPX4 in colon tissues. The results indicated that hucMSC-Ex treatment upregulated the Nrf2/HO-1/GPX4 pathway (Figure 4I-L and Figure 4N-P). Finally, an additional evaluation was conducted to ascertain the impact of hucMSC-Ex on the Nrf2/HO-1/GPX4 axis in different RAW264.7 phenotypes. Western blot results demonstrated increased Nrf2/HO-1/GPX4 expression following hucMSC-Ex treatment (Figure 4M and Figure 4Q-S). Collectively, these results indicated that hucMSC-Ex alleviates inflammation and oxidative stress by upregulating the Nrf2/HO-1/GPX4 axis.

Figure 4 Human umbilical cord mesenchymal stem cell-derived exosomes upregulate the nuclear factor-erythroid 2 related factor 2/heme oxygenase-1/glutathione peroxidase 4 axis to reduce inflammation and oxidative stress. A-C: Routine whole blood parameters as indicators of systemic inflammation; D and E: Serum alanine aminotransferase and aspartate aminotransferase levels as markers of liver function; F: Hematoxylin-eosin staining of mouse spleen (5 ×, scale bar = 400 μm) and liver tissues (30 ×, scale bar = 200 μm); G and H: Activities of the antioxidant markers glutathione and superoxide dismutase in colon tissues; I: Protein expression levels of the nuclear factor-erythroid 2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/glutathione peroxidase 4 (GPX4) axis in mouse colon tissues; J-L: Quantitative analysis of protein expression of Nrf2, HO-1, and GPX4 by Western blot; M: Western blot analysis of Nrf2, HO-1, and GPX4 expression in RAW264.7 cells after human umbilical cord mesenchymal stem cell-derived exosome (hucMSC-Ex) treatment; N-P: Messenger RNA expression levels of Nrf2, HO-1, and GPX4 in mouse colon tissues; Q-S: Quantitative analysis of protein expression of Nrf2, HO-1, and GPX4 in RAW264.7 cells treated with hucMSC-Ex. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: No significance; NC: Negative control; DSS: Dextran sulfate sodium; hucMSC-Ex: Human umbilical cord mesenchymal stem cell-derived exosomes; WBC: White blood cell; RBC: Red blood cell; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GSH: Glutathione; SOD: Superoxide dismutase; Nrf2: Nuclear factor-erythroid 2 related factor 2; HO-1: Heme oxygenase-1; GPX4: Glutathione peroxidase 4; M^PBS: Phosphate-buffered saline treatment; M^LPS: Lipopolysaccharide + interferon-γ treatment; M^IL-4: Interleukin-4 + interleukin-13 treatment; Ex: Exosomes; PBS: Phosphate-buffered saline.

HucMSC-Ex modulates the plasticity of intestinal macrophages

Intestinal-resident macrophages do not possess the capacity for self-sustainment; the continuous recruitment and maturation of circulating monocytes are required to occupy intestinal niches and maintain homeostasis of intestinal mucosal immunity. These macrophages originate from Ly6C-high (Ly6C^hi) monocytes, which migrate to the inflamed mucosa under the regulation of CCR2 in response to chemokines such as CCL2, CCL7, and CCL8. Upon entering the intestinal mucosa, CCR2⁺ Ly6C⁺ monocytes initiate a well-defined maturation process. They first acquire MHCII, followed by loss of Ly6C and CCR2 expression, and enhancement of F4/80, CD206, and CX3CR1 in mice. F4/80^high macrophages are considered intestinal resident macrophages. Therefore, we used FACS to assess immune cell development. The results demonstrated that, compared with the NC group, the number of inflammatory macrophages (Inf-Mφ; CD11b⁺ F4/80^low) recruited from the bone marrow was significantly increased in the DSS group. Conversely, Inf-Mφ significantly decreased in the hucMSC-Ex group compared with the DSS group. However, no significant differences were observed in resident macrophages (CD11b⁺ F4/80^high) across the groups (Figure 5A). In addition, newly infiltrated monocytes exhibit high Ly6C expression but lack MHCII expression (namely, Ly6C^hi MHCII^-). Subsequently, monocytes that gradually acquire MHCII expression are considered immature macrophages (Ly6C⁺ MHCII^low). Ultimately, immature macrophages lose Ly6C expression and exhibit characteristics of mature macrophages (Ly6C^- MHCII⁺). FACS data indicated that hucMSC-Ex promoted the differentiation of Ly6C⁺ MHCII^low immature macrophages into Ly6C^- MHCII⁺ mature macrophages with phagocytic and bactericidal functions (Figure 5A). Furthermore, we assessed the protein expression of F4/80, CD206, and CD86 in colon tissues using IF. The presence of F4/80⁺ CD206⁺ cells indicates pro-resolving macrophages, whereas F4/80⁺ CD86⁺ cells are indicative of pro-inflammatory macrophages. In line with the above findings, IF results demonstrated that hucMSC-Ex treatment significantly increased the number of pro-resolving macrophages and reduced the number of pro-inflammatory macrophages (Figure 5B-D). Taken together, these findings suggested that hucMSC-Ex played a protective role against DSS-induced colitis by facilitating the phenotypic transformation of macrophages from a pro-inflammatory to a pro-resolving state.

Figure 5 Human umbilical cord mesenchymal stem cell-derived exosomes modulate the plasticity of intestinal macrophages. A: Flow cytometric analysis of intestinal macrophages in colon tissues stained with anti-F4/80, anti-CD11b, anti-MHCII, and anti-Ly6C antibodies; B: Immunofluorescence (IF) staining of pro-resolving macrophages (F4/80⁺ CD206⁺) and pro-inflammatory macrophages (F4/80⁺ CD86⁺) by immunofluorescence (IF) in colon tissues (20 ×, scale bar = 200 μm); C: Quantification of F4/80⁺ CD206⁺ cells in colon tissues; D: Quantification of F4/80⁺ CD86⁺ cells in colon tissues. Data are presented as the mean ± SD. ^bP < 0.01. ^cP < 0.001. Each experiment was repeated three times independently (n = 8 mice per group). NS: No significance; NC: Negative control; DSS: Dextran sulfate sodium; hucMSC-Ex: Human umbilical cord mesenchymal stem cell-derived exosomes; CD: Cluster of differentiation; MHC: Major histocompatibility complex; Ly6C: Lymphocyte antigen 6C; Inf-Mφ: Inflammatory macrophages; Res-Mφ: Resident macrophages.

HucMSC-Ex promotes transformation of macrophage subsets

Previous studies have demonstrated that intestinal macrophages exhibit M1/M2 polarization with distinct functions in response to various stimuli. Upon activation by LPS and IFN-γ, macrophages polarize into M1-like macrophages, identified by the expression of iNOS, IL-1β, IL-6, TNF-α, CCL5, CXCL9, and MMP9. These pro-inflammatory factors lead to epithelial barrier disruption, tissue damage, and impaired wound healing. Upon activation by IL-4 and IL-13, macrophages can polarize into M2-like macrophages, characterized by expression of Mrc1, Arg1, IL-10, TGF-β, and IRF4. These M2-like macrophages play a crucial role in tissue repair and immune regulation. To elucidate the effect of hucMSC-Ex on macrophage polarization, we examined the mRNA expression levels of polarization markers by RT-qPCR, including IL-1β, IL-6, TNF-α, iNOS, Mrc1, Arg1, IL-10, and Ym1. The mRNA expression levels of the pro-inflammatory factors IL-1β, IL-6, TNF-α, and iNOS induced by DSS decreased after hucMSC-Ex treatment (Figure 6A). Conversely, compared with the DSS group, hucMSC-Ex treatment resulted in increased mRNA expression levels of the pro-resolving cytokines Mrc1, Arg1, IL-10, and Ym1 (Figure 6B). To further investigate whether the therapeutic effect of hucMSC-Ex on colitis was due to alterations in macrophage phenotypes, we co-cultured hucMSC-Ex with macrophages of different phenotypes derived from RAW264.7 cells under various stimulation conditions. Subsequently, we assessed polarization markers using RT-qPCR. The results indicated that hucMSC-Ex downregulated the expression of M1 markers, including IL-1β, IL-6, TNF-α, and IL-12 (Figure 6C). Conversely, the expression of M2 markers, such as Mrc1, Arg1, IL-10, and TGF-β, was upregulated under IL-4 + IL-13 stimulation (Figure 6D).

Figure 6 Human umbilical cord mesenchymal stem cell-derived exosomes promote transformation of macrophage subsets. A and B: Relative mRNA expression levels of the inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase, Mrc1, Arg1, IL-10, and Ym1 determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR); C and D: Relative mRNA expression of the M1-related genes IL-1β, IL-6, TNF-α, and IL-12, and the M2-related genes Mrc1, Arg1, IL-10, and transforming growth factor-β determined by RT-qPCR in RAW264.7 cells stimulated with phosphate-buffered saline, lipopolysaccharide + interferon-γ, and IL-4 + IL-13 following human umbilical cord mesenchymal stem cell-derived exosome treatment. Data are presented as the mean ± SD. Each experiment was repeated three times independently (n = 8 mice per group). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: No significance; NC: Negative control; DSS: Dextran sulfate sodium; hucMSC-Ex: Human umbilical cord mesenchymal stem cell-derived exosomes; IL: Interleukin; mRNA: Messenger RNA; TNF: Tumor necrosis factor; iNOS: Inducible nitric oxide synthase; TGF: Transforming growth factor; M^PBS: Phosphate-buffered saline treatment; M^LPS: Lipopolysaccharide + interferon-γ treatment; M^IL-4: Interleukin-4 + interleukin-13 treatment; Ex: Exosomes; PBS: Phosphate-buffered saline.

HucMSC-Ex regulates macrophage phenotype via the JAK1/STAT1 signaling pathway

Activation of the JAK/STAT pathway significantly promotes inflammatory responses in IBD. The classical JAK/STAT signaling pathway plays a crucial role in macrophage polarization. Classical activation involves STAT1, whereas alternative activation involves STAT3, STAT5, and STAT6. Consequently, we focused on the JAK/STAT signaling pathway and assessed its activity following various stimuli in the presence or absence of hucMSC-Ex in mice with DSS-induced colitis. Western blot analysis demonstrated a decrease in the expression levels of JAK1 and p-STAT1 in response to hucMSC-Ex treatment (Figure 7A-D). An additional evaluation was conducted to ascertain the impact of hucMSC-Ex on JAK/STAT signaling activity in different phenotypes of RAW264.7 cells. The results demonstrated that the expression levels of JAK1 and p-STAT1 in M^LPS macrophages were significantly higher than those observed in M^PBS macrophages and M^IL-4 macrophages. Concurrently, Western blot analysis indicated a decrease in JAK1 and p-STAT1 expression following hucMSC-Ex treatment (Figure 7E-H). Furthermore, a p-STAT inhibitor was used to inhibit STAT1 in the M^PBS and M^LPS groups followed by hucMSC-Ex administration. Subsequently, the expression of macrophage polarization markers was examined. The results demonstrated that inhibition of p-STAT1 completely counteracted the effect of hucMSC-Ex on macrophage polarization, indicating that the function of hucMSC-Ex relied on modulation of the STAT1 signaling pathway (Figure 7I-L). In addition, Western blot analysis further revealed that under LPS + IFN-γ stimulation, hucMSC-Ex treatment downregulated p-STAT1 in the M^LPS group, whereas inhibition of p-STAT1 entirely negated the effects of hucMSC-Ex on macrophages (Figure 7M and N). Taken together, these results indicated that hucMSC-Ex regulates macrophage phenotype by repressing the JAK1/STAT1 signaling pathway.

Figure 7 Human umbilical cord mesenchymal stem cell-derived exosomes regulate macrophage phenotype via the Janus kinase 1/ signal transducer and activator of transcription 1 signaling pathway. A-D: Western blot analysis of Janus kinase (JAK) 1, signal transducer and activator of transcription (STAT) 1, and phospho-STAT1 (p-STAT1) protein expression in colon tissues; E-H: Western blot analysis of JAK1, STAT1, and p-STAT1 protein expression in RAW264.7 cells treated with human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex); I-L: Determination of interleukin-1β, tumor necrosis factor-α, transforming growth factor-β, and Mrc1 mRNA expression by reverse transcription quantitative polymerase chain reaction after STAT1 inhibition followed by hucMSC-Ex administration; M and N: Determination of protein expression of p-STAT1 and STAT1 by Western blot after STAT1 inhibition followed by hucMSC-Ex administration. Data are presented as the mean ± SD. Each experiment was repeated three times independently (n = 8 mice per group). ^aP < 0.05. ^bP < 0.01. NS: No significance; NC: Negative control; DSS: Dextran sulfate sodium; hucMSC-Ex: Human umbilical cord mesenchymal stem cell-derived exosomes; JAK: Janus kinase; STAT: Signal transducer and activator of transcription; p-STAT: Phospho-signal transducer and activator of transcription; M^PBS: Phosphate-buffered saline treatment; M^LPS: Lipopolysaccharide + interferon-γ treatment; M^IL-4: Interleukin-4 + interleukin-13 treatment; Ex: Exosomes; PBS: Phosphate-buffered saline; IL: Interleukin; mRNA: Messenger RNA; TNF: Tumor necrosis factor; TGF: Transforming growth factor; STAT1 inh: Signal transducer and activator of transcription 1 inhibitor.

DISCUSSION

IBD, encompassing UC and Crohn’s disease, is characterized by chronic and debilitating intestinal inflammation. Patients suffer from various and lifelong symptoms due to persistent inflammation, with varying severity and treatment response[39-42]. Significant advances in therapeutic research have ushered in a new era of IBD treatment, including anti-TNF-α biologic agents, anti-IL-12/23 p40 biologics, anti-integrin biologics, a JAK inhibitor, and a sphingosine-1-phosphate receptor modulator[43,44]. However, adverse reactions and inconsistent responses have limited their application[45,46], highlighting the need for alternative therapies. In recent years, hucMSC-Ex has garnered considerable attention as a potential alternative. In this study, we investigated the effect of intraperitoneal injection of hucMSC-Ex on DSS-induced colitis. Based on our data, hucMSC-Ex exerts significant therapeutic effects on colitis. Given the capabilities of hucMSCs in immune regulation, tissue repair, and tissue regeneration[47], we explored the protective effect of hucMSC-Ex on the intestinal mucosal barrier. Furthermore, we observed that hucMSC-Ex administration influences intestinal immune responses by enhancing the immune response of M2-like macrophages and reducing that of M1-like macrophages in the lamina propria. Additionally, hucMSC-Ex treatment can enhance the antioxidant capacity of macrophages by upregulating the Nrf2/HO-1/GPX4 axis. Finally, we found that hucMSC-Ex can regulate macrophage phenotypic plasticity by downregulating the JAK1/STAT1 pathway. Our findings indicate that hucMSC-Ex is a promising candidate for IBD therapy, with the ability to protect the intestinal barrier and regulate immune responses.

Previous studies have shown that the emerging cell-free therapy hucMSC-Ex has a remarkable impact on repairing IBD. Mao et al[30] have demonstrated that hucMSC-Ex could markedly alleviate DSS-induced IBD in mice by increasing IL-10 and decreasing TNF-α, IL-1β, IL-6, iNOS, and IL-7[30]. Liu et al[48] have indicated that hucMSC-Ex alleviates IBD by regulating RACK1 O-GlcNAcylation modification in intestinal epithelial cells (IECs)[48]. Zhang et al[32] have showed that hucMSC-Ex can regulate lymphangiogenesis via the miR-302d-3p/VEGFR3/AKT axis to ameliorate IBD. Consistent with these studies, our findings demonstrated that hucMSC-Ex could alleviate DSS-induced colitis by downregulating the overall inflammatory response, reducing neutrophil infiltration, decreasing pro-inflammatory cytokines, and increasing anti-inflammatory cytokines.

The intestinal mucosal barrier includes the chemical barrier of the mucus layer, the mechanical barrier of the IEC layer, and the immune barrier of the LP[49]. The mucus layer acts as the first barrier, preventing direct contact between intestinal bacteria and IECs. The mucus layer consists of mucins, antimicrobial peptides, and secreted immunoglobulin A. Among them, Muc2, which is synthesized by goblet cells, is the most important component[50,51]. The mechanical barrier, consisting of the key proteins claudin-1, occludin, ZO-1, and E-cadherin, is essential for maintaining the integrity and function of the intestinal barrier[52]. In IBD, disruption of the intestinal mucosal barrier includes mechanical barrier damage, reduced mucus layer thickness, and dysfunction of goblet and Paneth cells, which subsequently leads to increased intestinal permeability[53,54]. Our results indicated that hucMSC-Ex treatment significantly increases the expression of Muc2 and mechanical barrier molecules and decreases intestinal permeability. These results fully substantiate the reparative effect of hucMSC-Ex on intestinal damage.

Dysfunction and subpopulation dysbiosis of T cells disrupt the balance of the intestinal immune barrier and promote the development of IBD. Other studies have shown that hucMSC-Ex treatment can alleviate experimental colitis by regulating the Th2/Th17 balance and the regulatory T cell/Th17 balance in the spleen and mesenteric lymph nodes[35,55,56]. As another important immune cell population in the immune barrier, macrophages play a crucial role in establishing and maintaining intestinal homeostasis. A large body of evidence has indicated that intestinal macrophages are involved in cytokine and chemokine production, macrophage extracellular trap formation, regulation of immune cell interactions, modulation of immune cell–epithelial cell crosstalk, and activation of phagocytic and bactericidal functions[57]. Disruption of intestinal homeostasis leads to significant changes in the composition of the intestinal macrophage pool during IBD, characterized by massive infiltration of pro-inflammatory macrophages[58]. Accordingly, we first observed that immature inflammatory macrophages (Inf-Mφs) were significantly increased in the DSS group, whereas their numbers decreased in the hucMSC-Ex group. When monocytes enter the intestinal mucosa, they initiate a maturation process and eventually transform into mature macrophages. This entire maturation process is termed the "monocyte waterfall" and typically takes approximately 5-7 days[57,59,60]. Lu et al[58] have emphasized that intestinal macrophage maturation is arrested at the immature stage during IBD, and that MCPIP1 deficiency is involved in this process via the ATF3-AP1S2 axis. In this study, we observed that hucMSC-Ex treatment could reverse the restricted maturation process of macrophages in the DSS group. Moreover, macrophage phenotype shifted from pro-inflammatory M1-like macrophages to anti-inflammatory M2-like macrophages, both in vivo and in vitro. Furthermore, we also discovered that hucMSC-Ex treatment can enhance antioxidant capacity by upregulating the Nrf2/HO-1/GPX4 pathway in macrophages, which is supported by other studies demonstrating that activation of the Nrf2/HO-1 pathway leads to reduced inflammation and oxidative stress[61]. However, it should be noted that this study did not involve direct intervention in the Nrf2/HO-1/GPX4 axis; therefore, the upregulation of this axis by hucMSC-Ex to enhance macrophage antioxidant capacity remains an observed phenomenon. Future studies involving pathway intervention will be needed to establish a causal relationship.

The JAK/STAT signaling pathway is an evolutionarily conserved transmembrane signal transduction mechanism that enables cells to communicate with the external environment[62]. Once receptors bind to ligands, JAKs undergo receptor tyrosine phosphorylation and recruit STATs. Subsequently, STATs undergo tyrosine phosphorylation and form STAT dimers. These STAT dimers then enter the nucleus and promote the transcription of target genes[62,63]. Previous studies have indicated that the JAK-STAT pathway is implicated in the onset and progression of various autoimmune diseases, such as rheumatoid arthritis, Alzheimer’s disease, and IBD. In addition, the JAK/STAT pathway plays a crucial role in the differentiation of monocytes into macrophages and in macrophage polarization, and is also involved in the progression of IBD[64-66]. Wang et al[67] reported that the expression of JAK1 and STAT1 was upregulated in the colon and lung tissues of mice with DSS-induced colitis. In that study, cimifugin ameliorated UC-associated lung injury by inhibiting the JAK1/STAT1 pathway and macrophage M1 polarization. In our study, we found that hucMSC-Ex treatment could downregulate the expression of JAK1 and p-STAT1 in colitic mice. Furthermore, we examined the effects of hucMSC-Ex on the JAK1/STAT1 pathway in macrophage polarization in vitro. The protein expression of JAK1 and p-STAT1 was significantly reduced in M^LPS macrophages treated with hucMSC-Ex. This is consistent with the findings of a study showing that TSG-6 protein secreted by hucMSCs mitigates the condition of Crohn’s disease patients by reducing M1 macrophage polarization through inhibition of JAK/STAT1 phosphorylation[68]. Interestingly, hucMSC-Ex treatment decreased total STAT1 but slightly increased p-STAT1 expression under basal conditions, whereas the opposite effect was observed under LPS + IFN-γ stimulation. Studies have shown that pretreatment of MSCs with IFN-γ, TNF-α, and IL-1β can enhance the therapeutic effect of MSCs on UC. Therefore, we speculate that the inflammatory environment can enhance the protective effect of hucMSC-Ex. Taken together, these experimental data clarify that hucMSC-Ex may modulate macrophage phenotype via the JAK1/STAT1 pathway.

CONCLUSION

In summary, we investigated the protective effects and underlying mechanisms of hucMSC-Ex on DSS-induced colitis and provided evidence that hucMSC-Ex is effective in the treatment of DSS-induced colitis. Our findings indicated that hucMSC-Ex can alleviate overall inflammation in mice, repair the intestinal barrier, enhance antioxidant capacity, and maintain immune homeostasis. Mechanistically, hucMSC-Ex altered macrophage plasticity by downregulating the JAK1/STAT1 pathway. In conclusion, this study not only provides guidance for further research on the molecular mechanisms of IBD but also offers a theoretical basis for targeted therapy of IBD.