Systems-level investigation of the anxiolytic gut-brain interactions induced by paraprobiotic Lactobacillus brevis SBC8803 in zebrafish.

INTRODUCTION: Anxiety disorders are among the most prevalent mental health conditions worldwide, and interest in psychobiotics-live or inactivated microorganisms that beneficially modulate the microbiota-gut-brain axis-is increasing. Heat killed Lactobacillus brevis SBC8803 enhances serotonin (5 hydroxytryptamine; 5 HT) signaling and ameliorates stress-related phenotypes in mammals, although the gut-brain pathways mediating these effects remain incompletely defined. Here, we investigated the anxiolytic effects and underlying molecular mechanisms of oral SBC8803 administration in adult zebrafish.

METHODS: Adult male AB-strain zebrafish were fed a diet containing heat killed SBC8803 for 4 weeks, and anxiety-like behavior was evaluated using the novel tank test. To explore the underlying mechanisms, we performed brain RNA sequencing and V3-V4 region of 16S rRNA amplicon sequencing of intestinal contents, followed by integrative multi omics analyses, including Gene Set Variation Analysis (GSVA) combined with DIABLO-based data integration and residual correlation analysis.

RESULTS: SBC8803-treated fish exhibited a shorter latency to enter the upper half of the tank and more frequent entries into this region, consistent with reduced anxiety-like behavior. Brain transcriptomic profiling identified differentially expressed genes and enrichment of serotonin receptor, CREB, and oxytocin signaling pathways, suggesting enhanced monoaminergic and plasticity-related signaling. Microbiome functional prediction indicated SBC8803-associated shifts in lipid and vitamin metabolism, including pathways related to riboflavin (vitamin B2) and tryptophan. GSVA combined with DIABLO-based data integration revealed coordinated changes between microbial metabolic and brain signaling pathways, consistent with a vitamin B-serotonin-anti-inflammatory axis linking gut metabolism to neural regulation. Furthermore, residual correlation analysis showed innate gut-brain coordination independent of the SBC8803 effect, such as the coupling between brain arachidonic acid and gut histidine metabolism.

DISCUSSION: These findings support the biological validity of the SBC8803 administration-associated interactions observed in the multi-omics analyses. These findings suggest that the paraprobiotic SBC8803 may exert anxiolytic-like effects in zebrafish and reshape gut-brain network states at behavioral, microbial, and transcriptomic levels, providing a potential mechanistic framework for considering heat killed SBC8803 as a candidate psychobiotic for anxiety-related conditions.