Exosomes Derived from Exercise-Trained Mice Bone Marrow Mesenchymal Stem Cells Improve Wound Healing by Inhibiting Macrophage M1 Polarization

Oral Sciences Rheumatology and Immunology Immunology Bioengineering Sports Medicine Cell Biology Biochemistry Life Sciences Medicine
bone marrow mesenchymal stem cells exosome treadmill exercise macrophage polarization M1 wound healing inflammation

I. Academic Background and Research Significance

Wound healing is a highly complex physiological process that plays a crucial role in tissue regeneration, repair, and immune regulation. However, poor healing of chronic wounds is clinically widespread, severely impacting patients’ quality of life and increasing medical and socioeconomic burdens. The inflammatory response, being the first stage of wound healing, is pivotal in determining the quality of subsequent healing processes. Excessive or prolonged inflammation can lead to delayed healing and scar formation. Macrophages, as key regulators of the immune microenvironment, are central in modulating inflammation, tissue repair, and scar formation. Among them, M1-type macrophages are inclined towards pro-inflammatory responses, and their overactivation aggravates inflammation and hinders normal repair. Therefore, effectively regulating macrophage polarization has become a crucial scientific challenge in improving chronic wound healing.

In recent years, Mesenchymal Stem Cells (MSCs), due to their self-renewal and multidirectional differentiation potential, as well as their outstanding immunoregulatory and tissue repair capabilities, have become a hotspot in regenerative medicine and inflammation regulatory research. Increasing evidence shows that the paracrine effect of MSCs, especially through the secretion of exosomes, plays a dominant role in promoting tissue regeneration and immunoregulation. Exosomes not only significantly reduce the risk of immune rejection and transplantation-related risks associated with cell therapy but also serve as important mediators of extracellular signaling and intercellular communication due to their rich payload of proteins, lipids, and non-coding RNAs. Previous studies have found that MSC-derived exosomes can regulate macrophage polarization towards the anti-inflammatory M2 phenotype to promote wound healing, but the mechanisms and applications of using MSC exosomes to inhibit M1 polarization still require further exploration.

On the other hand, moderate exercise has long been proven to have positive effects on systemic immunity, metabolism, and tissue repair, being able to regulate both the number and function of MSCs, enhancing their proliferation and migration abilities. Some studies have shown that MSCs pretreated with drugs or physiological stimuli can enhance their paracrine and therapeutic activities, but the influence of exercise intervention on the secretory function, immune regulation, and tissue repair promotion of MSCs and their exosomes has not yet been systematically reported in original data.

Thus, this study focuses on whether moderate aerobic exercise can fundamentally enhance the exosome secretion and function of Bone Marrow-derived Mesenchymal Stem Cells (BMSCs), and whether it can, by inhibiting macrophage M1 polarization, effectively alleviate inflammation and accelerate wound healing. This scientific question not only enriches the theory of the exercise–stem cell–immunoregulation cascade mechanism but also provides cutting-edge experimental and theoretical foundations for the development of new cell-free tissue repair therapeutic strategies.

II. Source of the Paper and Author Introduction

The study is titled “Exosomes derived from bone marrow-derived mesenchymal stem cells of exercise-trained mice improve wound healing by inhibiting macrophage M1 polarization.” The first author is Jiling Qiu, with other authors including Yifan Zhao, Yingyi Chen, Yanxue Wang, Juan Du, Junji Xu, Lijia Guo, and corresponding author Yi Liu. All authors are from the Beijing Stomatological Hospital, Capital Medical University, the Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, the Laboratory of Tissue Regeneration and Immunology, and the Department of Orthodontics. The paper was published in 2025 by the authoritative academic journal of Oxford University Press.

III. Detailed Research Procedure

1. Experimental Design and Research Subjects

The study centers on topics such as “BMSC-derived exosomes, exercise intervention, macrophage polarization, inflammation, and wound healing,” employing systematic in vivo and in vitro experiments with mouse models as the main subjects. The research workflow covers animal exercise intervention, BMSC isolation and culture, exosome extraction and functional identification, in vitro macrophage function experiments, molecular mechanism analysis, in vivo skin wound models and cell ablation, as well as tissue pathology and molecular biology assays at multiple levels.

Main Experimental Steps:

(1) Establishment of Exercise Mouse Model

  • Subjects: 12 C57BL/6J SPF-grade male mice, 6 weeks old, about 20g.
  • Grouping: Randomly divided into a control group (5 sedentary mice) and an exercise group (5 treadmill-trained mice), housed independently with free access to food and water.
  • Exercise Program: The exercise group underwent gradually intensified treadmill training for 6 consecutive weeks: 7 m/min for the first 2 weeks, then 9 m/min for the next 4 weeks, 30 minutes per session per day.

(2) Isolation and Culture of BMSCs

  • After euthanasia, bone marrow was collected from femurs and tibias, diluted with PBS and centrifuged to collect bone marrow cells.
  • Cells were cultured in α-MEM medium, passaged at 80% confluence, and the third passage was used for experiments.
  • Cell proliferation detected by CCK-8; apoptosis detected by Annexin V-FITC flow cytometry.

(3) Extraction and Identification of Exosomes

  • Conditioned media was used to induce BMSCs to secrete exosomes, which were extracted by gradient ultracentrifugation.
  • Purified exosomes were characterized by transmission electron microscopy (TEM), particle size and distribution analysis (multi-angle particle size analysis), and Western blot for specific proteins: CD9 and CD81 as positive markers, Calnexin as a negative control.

(4) In Vitro Macrophage Extraction and M1 Polarization Model

  • 10-week-old male C57BL/6J mice received intraperitoneal injection of 4% thioglycolate solution to recruit macrophages; peritoneal lavage fluid was collected 3 days later and centrifuged to harvest cells.
  • M1 polarization induction: four groups (normal control, LPS [500 ng/ml] group, LPS + control exosome group, LPS + exercise exosome group), each treated for 24 hours.

(5) Exosome Uptake Detection

  • Exosomes were labeled with the Cy5-E SE fluorescent probe and co-incubated with macrophages for 6 hours; uptake confirmed by confocal microscopy.

(6) Detection of Inflammatory and Polarization-related Molecules

  • qPCR to detect expression of inflammatory factors (TNF-α, IL-6, IL-1β) in macrophages.
  • Flow cytometry to analyze the proportion of M1 (CD86+) cells.
  • Western blot to detect the expression and phosphorylation status of signaling proteins p65, p-p65, p38, and p-p38.

(7) Full-Thickness Skin Wound Healing Model in Mice

  • 38 male mice, 8 weeks old; after dorsal hair removal, a circular full-thickness wound (10mm diameter) was made; mice divided into control, PBS, control exosome (300μg), and exercise exosome (300μg) groups; local injection administered.
  • Some mice received intravenous Clophosome-A liposome treatment for macrophage depletion, to investigate the role of macrophages in the repair process.

(8) Tissue Pathology and Molecular Analysis

  • H&E and Masson staining to observe inflammatory cell infiltration and collagen synthesis.
  • Immunohistochemistry (IHC) to detect inflammatory factors (TNF-α, IL-6, IL-1β).
  • Immunofluorescence (IF) double staining to detect macrophages (F4/80+) and M1-type (iNOS+) cells.

2. Data Analysis Methods

  • GraphPad Prism 9.4 statistical software was used.
  • Data were expressed as mean ± SD, with one-way ANOVA for comparisons among multiple groups; statistical significance considered at p≤0.05.

IV. Main Research Results and Logic

1. Exercise Enhances BMSC Proliferation and Exosome Secretion

  • Exercise group BMSCs showed significantly enhanced proliferation, with no significant difference in apoptosis.
  • TEM revealed both exercise and control group exosomes were vesicular, ~110 nm in diameter, with identical particle size distributions (60–160 nm) and no obvious structural differences.
  • Western blot: Both groups’ exosomes highly expressed CD9 and CD81, but no Calnexin detected, indicating controlled purity.
  • Protein quantification and DLS analysis confirmed that exercise notably increased exosome yield per unit cell.

2. Exercise Exosomes Inhibit Macrophage M1 Polarization (In Vitro)

  • Confocal imaging confirmed exosomes could be taken up by macrophages.
  • qPCR and flow cytometry: Macrophages treated with exercise exosomes had significantly lower expression of pro-inflammatory factors (TNF-α, IL-6, IL-1β) and M1 marker CD86 positivity than controls.
  • Western blot: LPS group showed increased p65/p38 phosphorylation; control exosomes could downregulate this pathway, with exercise exosomes resulting in a more pronounced downregulation, indicating effective M1 polarization inhibition by blocking p65/p38 activation.

3. Exercise Exosomes Promote Wound Healing (In Vivo)

  • Wound area photographs taken at days 1, 3, 6, and 9 post-injury showed the exercise exosome group healed fastest and better than controls.
  • H&E and Masson staining showed the exercise exosome group had reduced inflammatory cell infiltration, restored dermal structure, and denser, thicker collagen fibers.

4. Exercise Exosomes’ Healing Effect is Dependent on Macrophages

  • In the macrophage-depleted (F4/80 negative) group, exercise exosomes could no longer promote wound healing, indicating that their mechanism is dependent on the presence and functional regulation of macrophages.

5. Exercise Exosomes Inhibit Inflammation and M1 Polarization (In Vivo)

  • IHC assay (day 4 post-injury): The expression of TNF-α, IL-6, and IL-1β in wound edge tissues was significantly lower in the exercise exosome group than in the control exosome group.
  • IF assay: The number of F4/80+iNOS+ (M1-type) double-positive macrophages was lowest in the exercise exosome group, with the most notable improvement in the inflammatory microenvironment.

V. Conclusions, Academic and Application Value

This study systematically reveals that moderate exercise intervention significantly enhances BMSC exosome secretion and boosts their immunoregulatory activity. As an efficient cell-free therapeutic strategy, exercise-derived exosomes can inhibit macrophage M1 polarization, reduce inflammatory factor levels, significantly improve the tissue inflammatory microenvironment, and accelerate full-thickness skin wound healing. The main mechanisms include blockade of the p65/p38 signaling pathway and suppression of the inflammatory cascade. The dependency of this effect on the presence of macrophages was further validated by ablation experiments, providing innovative evidence for understanding the exercise–stem cell–immunity–repair regulatory network.

VI. Research Highlights and Innovations

  1. Interdisciplinary Innovation: This study closely integrates exercise physiology, stem cell biology, immunology, regenerative medicine, and, for the first time, systematically proposes that exercise can reshape the function of MSC-derived exosomes.
  2. Rich Mechanistic Levels: Not only does it reveal the effect of exercise on MSC and exosome yield, but it also delves into their multi-level mechanisms of regulating macrophage polarization, molecular signaling, and wound repair.
  3. Rigorous Technical Route: Utilizing animal treadmill training, high-speed ultracentrifugation for exosome extraction, and a triad of flow cytometry/confocal/pathology detection, the study validates its hypothesis from multiple perspectives and layers.
  4. First Proposal of Exosome-mediated Exercise Immunomodulation: Paves a new direction for the health-promoting and translational regenerative applications of exercise.
  5. Highly Translatable Application Prospects: Exercise exosomes can serve as a novel cell-free tissue repair material, potentially overcoming the ethical and safety barriers of cell transplantation, and promoting the integration of precision immunoregulation and tissue repair.

VII. Other Important Information

  • Funding Support: This project was funded by the Beijing Municipal Administration of Hospitals’ Clinical Medicine Development Special Fund, the National Natural Science Foundation of China, and the Innovation Team Project of Beijing Stomatological Hospital, Capital Medical University.
  • Conflict of Interest: The authors declare no potential conflicts of interest.
  • Data Access: Relevant original data can be reasonably requested from the corresponding author.

VIII. Research Limitations and Outlook

Although this study unveils the mechanisms by which exercise exosomes affect macrophage polarization and wound repair, further exploration is required regarding their specific exosomal cargo, the profile of bioactive substances, and the regulatory crosstalk with other immune cells. In addition, future studies should further assess their safety and efficacy in large animals and clinical populations to provide more robust scientific evidence for translational applications.

IX. Comprehensive Evaluation

Jiling Qiu and colleagues, by utilizing a series of scientifically rigorous animal models and molecular biology techniques, have for the first time linked exercise, stem cell exosomes, and immunoregulation, broadening the research perspective in the wound repair field. Their conclusions not only provide new mechanistic insights for basic science but also showcase promising prospects for the application of cell-free exosome therapies. The study content is solid, the workflow rigorous, the data well-supported, and the innovation is outstanding, with important theoretical and practical significance for accelerating wound healing, intervening in chronic inflammation, and advancing cell-free regenerative therapies.