Probiotic-Derived Extracellular Vesicles Mediate the Neuroprotection of Walnut Green Husk Polysaccharide via the 5-HT/AQP1/Wnt Signaling Axis in Hippocampus

Background : Ochratoxin A (OTA), a prevalent and potent neurotoxic mycotoxin in the global food chain, crosses the blood-brain barrier to impair hippocampal neurogenesis and cause cognitive deficits. There is an urgent need to develop effective interventions against this significant neurological threat. Walnut green husk polysaccharide (WGP), a natural component with prebiotic potential, represents a promising candidate, but its protective mechanisms against OTA neurotoxicity, particularly through the gut-brain axis, remain entirely unknown. Results : This study demonstrates that WGP supplementation alleviates OTA-induced cognitive impairment and restores hippocampal neurogenesis in mice. Hippocampal transcriptomics identified that WGP reversed OTA-induced upregulation of Aquaporin 1 (AQP1) and suppression of the Wnt/β-catenin pathway. 16S rRNA sequencing revealed that WGP restored gut microbiota homeostasis, specifically rescuing the depleted probiotics Lactobacillus johnsonii and Akkermansia muciniphila. The gut microbiota's essential role was confirmed via fecal microbiota transplantation and antibiotic ablation. Direct supplementation with these probiotics or their isolated extracellular vesicles (EVs) recapitulated WGP's neuroprotection. Untargeted metabolomics pinpointed the serotonergic (5-HT) pathway as central to this EV-mediated effect, and administration of 5-hydroxytryptophan (5-HTP) directly ameliorated cognitive deficits. In vitro, OTA exposure in hippocampal neurons recapitulated AQP1/Wnt dysregulation. Mechanistically, 5-HT alleviated cytotoxicity by activating protein kinase C gamma (PKCγ), which led to the inhibition of AQP1 and the reactivation of Wnt/β-catenin signaling. Conclusions : This study delineates a novel gut-brain axis pathway wherein WGP alleviates OTA-induced cognitive impairment by remodeling the gut microbiota to augment probiotic-derived EVs. These EVs boost 5-HT signaling, which activates hippocampal PKCγ to inhibit AQP1, thereby reactivating the neurogenic Wnt/β-catenin pathway and promoting cognitive recovery. Our findings not only reveal a precise mechanism for dietary intervention against mycotoxin toxicity but also highlight probiotic-derived EVs and the hippocampal 5-HT/AQP1/Wnt axis as promising therapeutic targets.