Insight into the mechanism of Xiao–Chai–Hu–Tang alleviates irinotecan-induced diarrhea based on regulating the gut microbiota and inhibiting Gut β-GUS.

• Xiao–Chai–Hu–Tang attenuates irinotecan-induced diarrhea by mediating gut microbiota. • Xiao–Chai–Hu–Tang attenuates irinotecan-induced diarrhea by inhibiting gut β-GUS. • Xiao–Chai–Hu–Tang significantly reduced the concentration of SN-38 in the gut. • Xiao–Chai–Hu–Tang active ingredient baicalein selectively inhibited EcGUS activity. Irinotecan (CPT-11, Camptosar^@) is a first-line drug for metastatic colorectal cancer. CPT-11-induced diarrhea, which is closely related to the concentrations of β-glucuronidase (β-GUS) and SN-38 in the gut, largely limits its clinical application. Herein, Xiao–Chai–Hu–Tang (XCHT), a traditional Chinese formula, was applied to mitigate CPT-11-induced toxicity. This study initially explored the mechanism by which XCHT alleviated diarrhea, especially for β-GUS from the gut microbiota. First, we examined the levels of the proinflammatory cytokines and the anti-inflammatory cytokines in the intestine. Furthermore, we researched the community abundances of the gut microbiota in the CPT-11 and XCHT-treated mice based on 16S rRNA high-throughput sequencing technology. Meanwhile, the level of SN-38 and the concentrations of β-GUS in intestine were examined. We also resolved the 3D structure of β-GUS from gut microbiota by X-ray crystallography technology. Moreover, we used virtual screening, SPR analysis, and enzyme activity assays to confirm whether the main active ingredients from XCHT could selectively inhibit β-GUS. In XCHT-treated mice, the levels of the proinflammatory cytokines decreased, the anti-inflammatory cytokines increased, and the community abundances of beneficial Firmicutes and Bacteroidota improved in the gut microbiota. We also found that the concentrations of β-GUS and the level of SN-38, the major ingredient that induces diarrhea in the gut, significantly decreased after coadministration of XCHT with CPT-11 in the intestine. Additionally, we revealed the structural differences of β-GUS from different gut microbiota. Finally, we found that EcGUS had good affinity with baicalein and meanwhile could be selectively inhibited by baicalein from XCHT. Overall, XCHT could relieve the delayed diarrhea induced by CPT-11 through improving the abundance of beneficial gut microbiota and reduced inflammation. Furthermore, based on the three-dimensional structure, baicalein, especially, could be used as a candidate EcGUS inhibitor to alleviate CPT-11-induced diarrhea. [Display omitted] [ABSTRACT FROM AUTHOR]