Tongxie Yaofang ameliorates IBS-D by targeting the gut microbiota-derived tryptophan metabolites and AhR signaling axis

Background and aims Tongxie Yaofang (TXYF) is a classic prescription for IBS-D with liver depression and spleen deficiency, with its therapeutic mechanisms requiring further elucidation. This study investigated the modulatory effects of TXYF on the gut microbiota and microbiota-derived metabolism in an IBS-D rat model to elucidate the underlying mechanisms. Methods HPLC was employed to identify the main components of TXYF. An IBS-D rat model was replicated using a triple-factor approach that combined neonatal maternal separation, chronic restraint stress, and oral gavage of Sennae folium decoction. To elucidate the mechanisms underlying the effects of TXYF on IBS-D, the gut microbiota was assessed by 16S rRNA sequencing, and microbial metabolites were profiled via untargeted metabolomics. Furthermore, key regulatory factors were examined by immunohistochemistry (IHC), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blotting. Finally, fecal microbiota transplantation (FMT) was performed to validate the pathogenic role of the microbiota and the therapeutic potential of TXYF. Results TXYF significantly alleviated IBS-D symptoms in rats, including diarrhea and abdominal pain, and improved both depressive-like behavior and intestinal barrier function. Treatment with TXYF increased the abundance of Bifidobacterium in the gut microbiota and promoted the microbial-related metabolic conversion of tryptophan (TRP) to 5-hydroxyindoleacetic acid (5-HIAA), indole-3-acetic acid (IAA), tryptamine, 5-hydroxytryptamine (5-HT), and 2-oxindole. These metabolites activated the aryl hydrocarbon receptor (AhR) signaling pathway, thereby inhibiting MLC phosphorylation and decreasing MLCK expression, and ultimately restoring intestinal barrier function. Furthermore, the FMT experiment demonstrated that the microbiota from TXYF-treated rats significantly ameliorated IBS-D by activating the AhR signaling pathway. Conclusion TXYF may alleviate IBS-D symptoms and restore barrier function by increasing the abundance of Bifidobacterium , restoring tryptophan metabolism, and activating the AhR pathway.