Key Takeaways & Executive Findings
- •• ATG5 overexpression in MSCs enhances autophagy and antioxidant capacity, improving their survival and therapeutic potential in inflammatory microenvironments. • MSCs-ATG5 promotes M2 macrophage polarization and suppresses IL-17/NF-κB signaling, reducing colitis severity in a mouse model. • Multiomics analysis reveals that ATG5-engineered MSCs modulate oxidative stress and anti-inflammatory metabolites like PGD2, offering a comprehensive mechanism. • This study provides a novel strategy to enhance MSC-based therapy for IBD by genetic engineering, addressing key limitations in clinical translation.
Abstract
Background The therapeutic efficacy of mesenchymal stem cells (MSCs) can be improved by enhancing their adaptation to the inflammatory microenvironment. Autophagy maintains MSCs functionality, and autophagy-related gene 5 (ATG5) mediates autophagy and regulates the biological functions and therapeutic efficacy of these cells. The aim of this study was to investigate the role of ATG5 in the antioxidant capacity and evaluate the therapeutic effect of ATG5-engineered MSCs for colitis treatment. Methods Cell viability was assessed using a Cell Counting Kit-8. The mRNA expression of autophagy-, antioxidant-, and polarization-related genes was determined through real-time quantitative polymerase chain reaction, and protein expression was analyzed via western blotting. Macrophage polarization markers were analyzed using flow cytometry. Multiomics approaches, including RNA transcriptome sequencing, untargeted metabolomics, and 16S ribosomal RNA microbiota analysis, were also used. Mice with dextran sulfate sodium-induced colitis were used to evaluate the therapeutic efficacy of MSCs. Results Preconditioning MSCs with hypoxia (1% O₂) and serum deprivation significantly enhanced autophagy and upregulated ATG5 expression. Adenovirus-mediated ATG5 overexpression in MSCs (MSCs-ATG5) enhanced their autophagic activity and antioxidant capacity, upregulated HMOX-1, SOD2, and CAT expression, and increased glutathione peroxidase and catalase enzymatic activity, while enhancing cell proliferation, without altering surface marker expression. Further, MSCs-ATG5 significantly promoted M2 macrophage polarization and regulated oxidative stress-related signaling pathways. Additionally, MSCs-ATG5-based therapy markedly ameliorated colitis disease signs in mice. Transcriptome analysis revealed that MSCs-ATG5 suppressed the IL-17/NF-κB inflammatory signaling pathway. This treatment also regulated levels of the anti-inflammatory metabolite prostaglandin D2 (PGD2) in colon tissues.
1. Introduction
Mesenchymal stem cells (MSCs) have potent immunomodulatory capacity, low immunogenicity, homing, and paracrine properties, and they are associated with abundant sources; thus, they have considerable potential for applications in immunotherapy and tissue repair [1]. These cells have been extensively investigated and exploited in diverse disease contexts, including autoimmune, cardiovascular, neurological, digestive, and orthopedic diseases [2–6]. However, the clinical translation of MSCs is still hindered by multiple critical challenges, including the lack of standardized preparation protocols, limited in vivo survival and persistence, unoptimized administration strategies, and inter-individual variability in therapeutic efficacy [7]. Therefore, strategies aimed at enhancing the in vivo survival and homing efficiency of MSCs are pivotal for boosting their therapeutic efficacy in clinical settings.
Inflammatory bowel disease (IBD) comprises a group of idiopathic, nonspecific inflammatory disorders of the intestinal tract, primarily ulcerative colitis (UC) and Crohn’s disease (CD). Its pathogenesis is predominantly driven by complex interplay among genetic factors, environmental triggers, intestinal microbiota dysbiosis, and immune dysregulation [5]. Currently, no curative therapy is available for IBD. MSCs have emerged as a promising therapeutic modality and have been extensively investigated in preclinical studies. However, their therapeutic efficacy is directly contingent on the intestinal oxidative stress microenvironment, which modulates their homing capacity and survival [8]. This microenvironment exhibits elevated local concentrations of proinflammatory cytokines and reactive oxygen species (ROS), directly inducing MSCs apoptosis or functional impairment [9]. Additionally, the hypoxic intestinal milieu constrains MSCs energy metabolism, further compromising their in vivo survival [10]. Thus, enhancing MSC adaptation to the intestinal inflammatory microenvironment is a promising strategy for improving the efficacy of MSC-based therapies for IBD.
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Yang Sun, Huiyan Sun, Tengfei Zhao, Yanyun Zhao, Fengjun Xiao, Lisheng Wang (2026). ATG5 overexpression enhances the therapeutic efficacy of mesenchymal stem cells in a mouse colitis model by augmenting anti-inflammatory and antioxidative mechanisms. Stem Cell Research & Therapy. https://doi.org/10.1186/s13287-026-05008-2
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Frequently Asked Questions
What is the role of ATG5 in mesenchymal stem cells?
ATG5 is an autophagy-related gene that mediates autophagy and regulates the biological functions and therapeutic efficacy of MSCs. Overexpression of ATG5 enhances autophagic activity and antioxidant capacity, improving MSC survival and function in inflammatory microenvironments.
How does ATG5 overexpression improve MSC-based therapy for colitis?
ATG5 overexpression in MSCs enhances their anti-inflammatory and antioxidative mechanisms, promoting M2 macrophage polarization, suppressing IL-17/NF-κB signaling, and regulating anti-inflammatory metabolites like PGD2, thereby ameliorating colitis symptoms in a mouse model.
What methods were used in this study?
The study employed cell viability assays, real-time PCR, western blotting, flow cytometry, RNA transcriptome sequencing, untargeted metabolomics, 16S rRNA microbiota analysis, and a DSS-induced colitis mouse model to evaluate the effects of ATG5-engineered MSCs.
What are the key findings of this research?
Key findings include that hypoxia and serum deprivation upregulate ATG5, ATG5 overexpression enhances MSC antioxidant capacity and proliferation, promotes M2 polarization, suppresses inflammatory signaling, and improves colitis outcomes in mice.
What is the significance of this study for IBD treatment?
This study provides a novel strategy to enhance MSC-based therapy for IBD by genetic engineering of ATG5, addressing limitations such as poor survival and efficacy in inflammatory microenvironments, and offering a potential approach for clinical translation.
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