Depression involves dysregulation of central and peripheral networks, with gut microbiota alterations increasingly implicated in pathogenesis. Trigonella foenum-graecum L. (fenugreek), a functional food and medicinal herb, has been used traditionally for neurological disorders, yet its specific neuroprotective constituents remain poorly characterized. This study evaluated the antidepressant potential of schaftoside, the predominant flavonoid of fenugreek seed flavonoids, and explored its mechanisms involving the microbiome-gut-brain axis in a chronic restraint stress (CRS) mouse model. Male Kunming mice were subjected to CRS and orally administered schaftoside (20, 50, and 125 mg kg-1) or fluoxetine (10 mg kg-1) for 28 days. Behavioral assessments (tail suspension, forced swim, novel object recognition, and Y-maze) demonstrated that schaftoside dose-dependently ameliorated depressive-like behaviors and cognitive impairments. It attenuated hippocampal neuronal damage and neuroinflammation, restoring hippocampal 5-HT, NE, and ACh levels. Intestinal barrier integrity was improved, evidenced by enhanced tight junction proteins (ZO-1, occludin) and mucin (MUC2) expression, alongside reduced colonic pro-inflammatory cytokines and elevated IL-10. 16S rRNA profiling revealed that schaftoside reshaped gut microbiota composition, notably suppressing stress-enriched Bacteroides and promoting butyrate-producing Butyricicoccus. Untargeted metabolomics identified restoration of tryptophan and α-linolenic acid metabolism pathways. Spearman correlation analysis revealed significant associations between specific gut genera, fecal metabolites, and host inflammatory/neurochemical markers. Hippocampal transcriptomics indicated enrichment of cAMP signaling, dopaminergic, and serotonergic synapse pathways. Fecal microbiota transplantation (FMT) from schaftoside-treated donors partially recapitulated the behavioral, anti-inflammatory, and intestinal barrier-protective effects in antibiotic-pretreated recipient mice, accompanied by restoration of the gut microbiota and metabolite profiles. These findings demonstrate that schaftoside, the major flavonoid of fenugreek seeds, exhibits promising antidepressant-like potential through modulation of the microbiome-gut-brain axis, supporting the functional food application of fenugreek-derived flavonoids for stress-related mood disorders.
