Abstract

Aims Chemotherapy-induced intestinal mucositis (CIM) is a common and severe side effect linked to disrupted intestinal stem cell (ISC) balance, though its molecular regulation remains unclear. The principal objective of this research was to elucidate the role of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) in intestinal stem cell balance and mucosal repair during CIM, with a specific focus on the Wnt/β-catenin pathway. Materials and methods This study used multi-omics and gene editing to elucidate ECSIT's role in intestinal stem cell balance and mucosal repair via the Wnt/β-catenin pathway. Multi-omics and Gene Set Enrichment Analyses (GSEA) identified ECSIT as a key CIM regulator. Key findings Chemotherapeutic drugs dose-dependently reduced ECSIT expression in intestinal epithelial cells. Clinical analysis revealed that ECSIT expression decreased with increasing pathological severity (normal > inflammation > adenocarcinoma). In intestinal epithelium-specific knockout mice, ECSIT deficiency worsened irinotecan (CPT11)-induced CIM and blocked β-catenin nuclear translocation. ECSIT stabilized the β-catenin complex, regulating Wnt target genes like Axis inhibition protein ( AXIN ) and Cyclin D1 ( CCND1 ); its knockout reduced Wnt signaling. Single-cell sequencing (scRNA-seq) revealed that ECSIT knockout reduced Lgr5 stem cells and increased inflammatory cell infiltration. Lgr5-specific inducible knockout and ECSIT complementation demonstrated that restoring ECSIT reversed β-catenin inhibition and improved CIM pathology. Significance This study clarified ECSIT's dual role in stabilizing β-catenin and sustaining Wnt signaling—regulating Lgr5 intestinal stem cell proliferation and differentiation, epithelial renewal, and immune balance. These findings offer insights into CIM pathogenesis and establish the basis for developing targeted therapy through ECSIT-Wnt axis regulation.