Garlic-Derived Exosome-Like Nanoparticles Enhance Gut Homeostasis in Stressed Piglets: Involvement of <i>Lactobacillus reuteri</i> Modulation and Indole-3-propionic Acid Induction

The occurrence of pediatric diarrhea is frequently associated with inflammatory responses, compromised barrier function, and dysbiosis in the gut. These conditions are commonly triggered by stressors, similar to postweaning diarrhea observed in piglets. Garlic-derived exosome-like nanoparticles (GELNs) hold the potential for ameliorating stress-induced diarrhea, yet supporting evidence remains scarce. Following the successful isolation of GELNs, this study employed weaned piglets as a model to evaluate the regulatory effects of GELNs on intestinal barrier integrity, mucosal inflammation, and the gut microbiota and its metabolites. Weaned Bama miniature piglets were orally administered phosphate buffer saline (PBS) or GELNs, and 1 week later, samples were collected following slaughter. Histological and molecular biological techniques were performed to examine intestinal structure, tight junction protein expression, mucin secretion, T lymphocyte infiltration, and the levels of pro-inflammatory cytokines. The composition of the gut microbiota was analyzed using 16S rRNA sequencing, while its derived metabolites were profiled via untargeted metabolomics. Subsequently, correlation analyses were performed to evaluate the associations between the microbiota and its derived metabolites, as well as between the microbiota and the key indicators of intestinal barrier function and cytokine levels in response to GELNs. The isolated GELNs exhibit typical exosome characteristics in size and morphology, alongside a rich content of proteins and RNAs. The incidence of diarrhea in weaned piglets was reduced with supplementation of GELNs at a dosage of 50 mg/kg body weight, compared to the control group. In addition, piglets receiving GELNs displayed an increase in mucin content within the tissues of the jejunum, ileum, and colon, a decrease in CD8⁺ T lymphocyte counts in the colon, and suppression of pro-inflammatory cytokines (IL-8 and TNF-α) levels in the mucosal layers of both the jejunum and ileum. Furthermore, 16S rRNA sequencing unveiled that GELNs reshaped the colonic microbiota in weaned piglets by augmenting beneficial bacteria, notably <i>Lactobacillus</i> and <i>Lactobacillus reuteri</i>, correlating strongly with diminished TNF-α protein levels and heightened mucin expression. Metabolite analysis demonstrated a significant increase in indole-3-propionic acid, derived from the gut microbiota, in piglets supplemented with GELNs. This increase was positively correlated with the abundance of <i>Lactobacillus</i> and <i>Lactobacillus reuteri</i> and negatively linked with the protein levels of IL-8 and TNF-α in the gut. In summary, our study demonstrates that GELNs mitigate stress-related intestinal mucosal inflammation and enhance mucin production in the gut of weaned piglets, which is potentially achieved through the optimization of gut microbiota composition, specifically by increasing the abundance of <i>Lactobacillus</i> and <i>Lactobacillus reuteri</i>, as well as via the induction of the anti-inflammatory microbial metabolite indole-3-propionic acid. The findings presented here provide essential groundwork for the future development of GELNs as a therapeutic strategy aimed at enhancing gut homeostasis disruption caused by stress in both weaned piglets and children.