Abstract
<title>Abstract</title> <p> <bold>Background:</bold> Depression is associated with disruptions of gut microbiota and host metabolism. However, the coordination of metabolic changes across the gut, circulation, peripheral immune organs, and brain remains poorly understood. We investigated whether chronic restraint stress (CRS) is accompanied by linked microbial and metabolic alterations along the gut-spleen-brain axis in mice. <bold>Methods:</bold> Male C57BL/6J mice were randomly assigned to control or three-week CRS groups. Depressive-like and anxiety-like behaviors were evaluated using a series of specialized behavioral tests: the sucrose preference, forced swim, tail suspension, and open-field tests. Fecal bacterial communities were profiled by 16S rRNA gene sequencing, and untargeted metabolomics were performed on feces, serum, spleen, and hippocampus. Differential-abundance analysis, pathway enrichment, weighted gene co-expression network analysis (WGCNA), and Spearman correlation analysis were used to integrate systematically microbial, metabolic, and behavioral indicators. <bold>Results:</bold> CRS reduced sucrose preference and open-field activity and increased immobility time in the forced swim and tail suspension tests. CRS altered gut microbial community structure without affecting alpha diversity. We identified 1,719 fecal, 642 serum, 200 splenic, and 145 hippocampal differential metabolites. Tryptophan metabolism was recurrently enriched across fecal and host compartments. Cross-tissue WGCNA identified blue and turquoise metabolite modules associated with behavioral measures. Correlation networks further linked tryptophan-related metabolites to differential bacterial genera and depressive-like behaviors, with Corynebacterium and Mucispirillum among the most highly connected microbial nodes. <bold>Conclusions:</bold> CRS was associated with gut microbial dysbiosis and coordinated, tissue-specific alterations in tryptophan-related metabolism along the gut-spleen-brain axis. These findings provide a cross-tissue framework for investigating metabolic communication in stress-related behavioral phenotypes. Targeted metabolite validation and causal experiments are required. </p>