Abstract

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. 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. 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.