Rhoifolin Alleviates Ulcerative Colitis by Targeting NMNAT1 and Activating SIRT1‐FOXO1 Signaling to Enhance ILC3s Effector Function

ABSTRACT Ulcerative colitis (UC) is a chronic inflammatory bowel disorder with limited therapeutic options, particularly those capable of selectively reinforcing intestinal epithelial barrier integrity while preserving immune homeostasis. Here, Rhoifolin (3, 10 mg/kg) dose‐dependently alleviates DSS and TNBS‐induced colitis in mice. Mechanistically, Rhoifolin selectively potentiates ILC3s effector function without affecting their proliferation, apoptosis, intestinal homing, developmental differentiation, or the distribution of CCR6 + , NKp46 + , and double‐negative ILC3s subsets. This functional enhancement promotes IL‐22 production, thereby reinforcing epithelial barrier integrity in colonic epithelial cells and organoids. Integrated transcriptomics and untargeted metabolomics identified nicotinamide salvage as a central metabolic program driving ILC3s functional enhancement. NMNAT1 was identified as a direct molecular target of Rhoifolin. Molecular docking and dynamic simulations pinpointed Leu155 as a critical residue mediating Rhoifolin‐NMNAT1 interaction, and mutation of Leu155 antagonized metabolic activation and IL‐22 induction. Consistently, Rhoifolin‐induced NAD+ elevation enhanced SIRT1 activity, leading to FOXO1 deacetylation and nuclear translocation. Further CUT&Tag‐qPCR demonstrated FOXO1 enrichment at Il22 promoter, driving IL‐22 transcription in ILC3s. Collectively, our findings identify Rhoifolin as a potent ILC3s effector function enhancer and uncover a previously unrecognized NMNAT1‐NAD+‐SIRT1‐FOXO1 axis linking immunometabolic and epigenetic regulation to intestinal barrier protection, highlighting ILC3s empowerment as a promising therapeutic strategy for UC.