Xiaoyao San exerts antidepressant effects via the gut microbiota-brain axis: An integrative fMRI and multiomics study

Depression is characterized by a dysregulated brain-gut axis. Xiaoyao San (XYS), a classic Traditional Chinese Medicine formula for soothing Liver and strengthening Spleen, is clinically effective in alleviating depression. However, the systems-level mechanisms by which XYS coordinates gut-brain communication to exert its antidepressant effects remain insufficiently understood. This study aimed to systematically elucidate the antidepressant mechanisms of XYS, with a focus on identifying a key gut-derived metabolic pathway that modulates prefrontal cortex (PFC) function. A mouse model of depression was established using isolated housing combined with chronic unpredictable mild stress (CUMS). Mice were treated with XYS at low, medium, and high doses or paroxetine. We employed a multimodal approach, integrating behavioral tests, resting-state functional magnetic resonance imaging (rs-fMRI), gut microbiota profiling (16S rRNA sequencing), serum metabolomics and PFC transcriptomics. To establish causal evidence, pseudo-germ-free mice received fecal microbiota transplantation (FMT) from donor mice treated with XYS, followed by comprehensive behavioral and biochemical assessments. XYS treatment significantly ameliorated depressive-like behaviors and restored functional connectivity within emotion-regulation brain networks. Multi-omics integration revealed that XYS reshaped the gut microbiota, which was associated with a reduction in systemic levels of kynurenine (KYN), a key tryptophan-derived metabolite. In the PFC, this decrease in KYN was accompanied by the normalization of aryl hydrocarbon receptor (AhR) signaling activity. Furthermore, GABAergic neurotransmission, mediated by γ-aminobutyric acid (GABA), was enhanced, as evidenced by upregulated expression of glutamate decarboxylase 1 (Gad1), gamma-aminobutyric acid type A receptor subunit alpha1 (Gabra1), increased GABA content, and elevated levels of key synaptic plasticity-related molecules, including brain-derived neurotrophic factor (BDNF), postsynaptic density protein-95 (PSD-95), and synaptophysin (SYN). Critically, FMT from XYS-treated donors recapitulated the antidepressant phenotype in recipient mice, directly implicating the gut microbiota in these therapeutic effects. This study demonstrates that XYS alleviates depression by orchestrating a gut-brain signaling cascade that converges on the PFC to enhance inhibitory synaptic transmission. These findings provide novel and causal mechanistic insights into the brain-gut modulatory action of XYS, offering a comprehensive framework for its therapeutic potential in treating depression.