Dietary Galacto-Oligosaccharides Attenuate Intestinal Inflammation and Mucosal Injury in TNBS-Induced Zebrafish Enteritis

Background: Inflammatory bowel disease (IBD) involves dysregulated mucosal immunity and compromised intestinal barrier integrity. Prebiotic oligosaccharides have emerged as immunomodulatory candidates, yet their sex-specific effects on piscine mucosal immunity remain poorly understood. Zebrafish (Danio rerio) offers a robust vertebrate model for dissecting intestinal immunopathology due to conserved innate and adaptive immune signaling.Methods: This study employed network pharmacology to predict GOS anti-colitis targets, followed by in vivo validation in a TNBS-induced zebrafish colitis model. Male and female zebrafish were fed 0.4% GOS or inulin for 35 days prior to intraperitoneal TNBS challenge. Intestinal histopathology was assessed by H&E and AB-PAS staining; mRNA expression of IL-1β, IL-6, TNF-α, IFN-γ1, and IL-10 was quantified by RT-qPCR.Results: GOS (96.62% purity) was biosynthesized via β-galactosidase BMG and showed favorable biocompatibility without growth impairment. Post-TNBS challenge, GOS significantly improved survival compared to inulin controls. Notably, sex-specific immunomodulation was observed: female zebrafish exhibited downregulated pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IFN-γ1), whereas males displayed marked IL-10 upregulation. Histologically, GOS restored goblet cell counts, reduced crypt damage, and recovered mucus secretion, indicating reinforced intestinal barrier function.Conclusion: These findings demonstrate that GOS exerts dual anti-colitis mechanisms through sex-biased cytokine regulation and mucosal barrier restoration in zebrafish, supporting its potential as an immunoprotective feed additive in aquaculture and a translational model for human IBD therapeutics.