Background: Gut microbiota dysbiosis is associated with diarrhea-predominant irritable bowel syndrome (IBS-D). Visceral hypersensitivity (VH) is a hallmark symptom. Methods: Twenty-five female rats were allocated to either a control group (sham stress, n = 5) or a stressed group exposed to a 10-day water avoidance (WA) stress protocol to induce VH (n = 20). After stress induction, stressed rats received a 4 week intervention with phosphate-buffered saline (PBS), Bifidobacterium animalis subsp. lactis BB-12 (P1), Lactiplantibacillus plantarum ATCC 14917 (P2), or their combination (P1P2). Control rats were administered PBS. Visceral hypersensitivity was assessed using abdominal withdrawal reflex (AWR) scores, and metabolic changes in plasma, liver, and distal colon were analyzed using proton nuclear magnetic resonance (1H NMR)-based metabolomics. Findings: Water avoidance stress induced VH, as evidenced by higher AWR scores (P < 0.05) and lower pain thresholds compared to controls (38.47 ± 3.78 versus 29.48 ± 2.52; P < 0.001). Probiotics significantly reduced AWR scores at 20 and 80 mmHg and increased pain thresholds in comparison with the WA + PBS group (P < 0.05). Metabolomic profiling revealed that WA + PBS rats showed significant dysregulation in energy, amino acid,one-carbon,and lipid metabolism.Notable changes included elevated sarcosine, acetate, taurine, glutamate, tryptophan, 2-hydroxybutyrate, urea, and allantoin, with reduced O-acetylcarnitine in plasma; increased succinate, myo-inositol, isoleucine, threonine, glutamine, betaine, pyroglutamate, and O-phosphocholine with decreased taurine in the liver; and various amino acids, ketone bodies, glucose, glycerol, and one-carbon metabolites in the colon (P < 0.001). Probiotic supplementation largely restored these metabolic changes, with clustering analyses supporting the normalization of metabolic signatures. Conclusions: Probiotic supplementation ameliorated VH and reversed IBS-D-associated metabolic disruptions, illustrating its therapeutic potential and providing insights into the underlying mechanisms.

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