Decoding the functional differentiation of herb pairs in Tong-Xie-Yao-Fang: effects on gut microbiota, intestinal barrier function, and Wnt signaling in IBS-D mice

Background Diarrhea-predominant irritable bowel syndrome (IBS-D) is a prevalent functional gastrointestinal disorder with limited therapeutic options. Tong-Xie-Yao-Fang (TXYF), a classical Chinese herbal formula, has shown clinical efficacy, but the respective contributions of its full formula and of its constituent herb pairs, “Tonifying Tu (Earth)” (TT) and “Suppressing Mu (Wood)” (SM), remain incompletely characterized, particularly with respect to the interplay among the gut microbiota, the intestinal barrier, and Wnt signaling. Methods An IBS-D mouse model was established by combined water avoidance stress and chronic restraint stress. Mice received TXYF, TT, or SM for 14 days. Therapeutic effects were assessed by body weight, diarrhea rate, abdominal withdrawal reflex (AWR), intestinal permeability (FITC-dextran), histopathology (H&E, PAS, SEM), and qPCR of Wnt-related genes. Fecal metagenomics with KEGG functional annotation was performed to profile microbial composition and metabolic pathways. Results TXYF, TT, and SM alleviated IBS-D symptoms, accompanied by improvements in villus height, crypt depth, and goblet cell numbers. Tight junction ultrastructure appeared preserved in TXYF and SM groups, alongside upregulated β-catenin and downregulated RNF43, while Lgr5 showed no significant changes. Metagenomics suggested that TXYF was associated with higher microbial diversity, lower abundance of the opportunistic pathogen Staphylococcus, and enrichment of beneficial genera (Lactobacillus, Bifidobacterium, Faecalibacterium), alongside shifts in Mammaliicoccus and Muribaculum. Herb-pair decomposition indicated differences that were most apparent at the functional level: TT was associated with TCA cycle and tryptophan metabolism enrichment (alongside Akkermansia gains), whereas SM was associated with flagellar assembly and chemotaxis enrichment. At the genus level, TT accounted for most of the large taxonomic shifts, whereas the changes associated with SM were smaller and largely concordant with those of TT. Conclusion TXYF alleviated IBS-D in association with modulation of the gut microbiota, restoration of intestinal barrier function, and potential involvement of the Wnt signaling pathway. Herb-pair decomposition suggests differences mainly at the metabolic-pathway level: TT was associated with beneficial metabolic-support shifts, whereas SM was associated with motility-related functional shifts, with the whole-formula effect numerically exceeding that of either herb pair alone.