Abstract

Irinotecan disrupts bile acid enterohepatic circulation by inhibiting hepatic bile acid synthesis/uptake and intestinal reabsorption, leading to conjugated bile acid accumulation in the colon. XCHT restores this homeostasis by upregulating key transporters ASBT and OST. • Irinotecan abnormally activates the FAK-RhoA/ROCK signaling pathway to degrade tight junction proteins (ZO-1, Occludin) and disrupt the intestinal barrier, while XCHT inhibits this pathway to maintain intestinal epithelial barrier integrity and rebalances Th1/Th2 differentiation to alleviate intestinal inflammation. • Cholestyramine mimics the therapeutic effect of XCHT by reducing the accumulation of bile acids in the intestine, confirming that bile acid imbalance is a key driving factor for CID. Background Irinotecan (CPT-11) is widely used for colorectal cancer treatment, with delayed-onset diarrhea as its primary side effect. Xiao-Chai-Hu-Tang (XCHT) has been clinically observed to alleviate chemotherapy-induced diarrhea, but its underlying mechanisms remain unclear. Purpose This study aimed to elucidate the regulatory mechanisms through which XCHT alleviated CPT-11-induced diarrhea using an integrated multi-omics approach. Methods The chemical components of XCHT were detected using UHPLC-QE-Orbitrap-MS, and 12 active components were quantified via UHPLC-QQQ-MS/MS to ensure quality stability. CPT-11-induced diarrheal mice were established to evaluate the therapeutic effects of XCHT. Differential metabolites in liver and intestinal tissues among control, CPT-11, and XCHT mice were analyzed by untargeted metabolomics, followed by molecular network analysis using IPA. Bile acids were quantified using targeted UHPLC-QQQ-MS/MS method. Proteomics of colon tissues identified differentially expressed proteins, with functional enrichment conducted via GO and KEGG. Western blot and flow cytometry were used to validate the potential biopathway. Results Total 1108 chemical components were identified in X