Mesenchymal Stem Cell Derived Exosomes Alleviates Hirschsprung‐Associated Enterocolitis by Inhibiting AKT Phosphorylation in Macrophages Through miR‐223

<b>Background:</b> Mesenchymal stem cells (MSCs) inhibit macrophage inflammatory response and alleviate intestinal inflammation. However, the role of MSCs in Hirschsprung-associated enterocolitis (HAEC) remains uncertain. This study aims to investigate the effects of MSCs on HAEC and the mechanisms related to macrophages and MSCs. <b>Methods:</b> Immunofluorescence was used to measure CD68 and p-AKT in colonic tissues of HSCR patients with HAEC. Ednrb^-/- mice was used as HSCR model. The proportion of colonic tissue macrophages in WT and Ednrb^-/- mice was assessed by flow cytometry. The colonic tissues injury was evaluated with HE staining and the survival curves of mice were recorded. In vitro, macrophage-induced enterocyte death was induced by lipopolysaccharide (LPS). MSCs, MSC derived exosomes, miR-223, or MK2206 were added to macrophages, and the levels of miR-223 in macrophages after exosome treatment were measured by RT-qPCR. Flow cytometry was used to assess enterocyte death, western blot was performed to measure p-AKT expression in macrophages, and enzyme-linked immunosorbent assay (ELISA) was used to detect IL-1β concentration in macrophage supernatants and serum of Edrnb ^<i>-/-</i> mice. <b>Results:</b> Increased expression of CD68 and p-AKT was observed in the colonic tissues of HAEC patients. Colonic instillation of MSCs derived exosomes significantly reduce the inflammatory score of colonic tissues and prolong the survival time of HAEC mice. In vitro, LPS-stimulated macrophages induce the phosphorylation of AKT and enterocyte death. Stimulation of macrophages with MSC-derived exosomes increased the content of miR-223. MSC-derived exosomes, miR-223 and MK2206 significantly reduce macrophage-induced enterocyte death, attenuated AKT phosphorylation in macrophages, and decreased IL-1β concentration in macrophage supernatants. <b>Conclusion:</b> Macrophages accumulate in colonic tissues during HAEC and inflammatory macrophages drive enterocyte death. MSCs derived exosomes reduce enterocyte death by suppressing AKT phosphorylation and IL-1β secretion via miR-223, and subsequently mitigate HAEC in mice. These findings suggest that MSC-derived exosomes, particularly those enriched in miR-223, may serve as a promising therapeutic strategy for the prevention or treatment of HAEC.