Xiao-Chai-Hu-Tang alleviates irinotecan-induced diarrhea via promoting bile acid reabsorption and decreasing focal adhesion kinase signaling revealed by metabolomics and proteomics.

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.

This study aimed to elucidate the regulatory mechanisms through which XCHT alleviated CPT-11-induced diarrhea using an integrated multi-omics approach. 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. Total 1108 chemical components were identified in XCHT, mainly including flavonoids, terpenoids and phenylpropanoids.

XCHT significantly improved diarrhea symptoms and reduced intestinal inflammation. Metabolomics revealed 117 and 53 differential metabolites in the liver and intestine, respectively, with both tissues showing alterations in bile acid metabolism. Targeted bile acid analysis showed that CPT-11 inhibited the synthesis and uptake of bile acids in the liver and enhanced bile excretion, resulting in an increase in bile acids in the gallbladder. Meanwhile, CPT-11 impaired the reabsorption of bile acids in the intestine, leading to a decrease in total bile acid levels, reduction of circulating bile acids, and accumulation of conjugated bile acids in the colon.

XCHT reversed these abnormal phenomena, restoring the intestinal bile acid homeostasis. Proteomics identified 86 differentially expressed proteins in colon tissues, with significant enrichment in 'Focal adhesion' pathway. Further verification indicated that CPT-11 abnormally activated the focal adhesion kinase (FAK) and its downstream RhoA/ROCK pathways, then reduced tight junction proteins ZO-1 and Occludin, damaging the intestinal barrier. XCHT inhibited this pathway, restored the expression of tight junction proteins, balanced Th1/Th2 differentiation, and alleviated intestinal inflammation.

Cholestyramine, a bile acid chelator, also alleviated CPT-11-induced diarrhea by reducing the accumulation of bile acids in the intestine, confirming the key role of bile acids in the treatment of CPT-11-induced diarrhea.

This study revealed that the combined administration of XCHT could restore bile acid reabsorption, reduced the accumulation of conjugated bile acids in the colon, inhibited the abnormally activated FAK-RhoA/ROCK pathway, improved intestinal epithelial barrier function and alleviate diarrhea.

These findings provide a new entry point for the treatment of chemotherapy-induced diarrhea (CID) and offer data support for the combined medication regimen of XCHT and CPT-11.