2′-Fucosyllactose and its metabolites propionate/butyrate suppress inflammation through a shared TLR4/p38 MAPK-dependent pathway in vitro and in vivo

2′-Fucosyllactose (2′-FL), the most abundant human milk oligosaccharide (HMO), exhibits potent anti-inflammatory properties, yet the underlying mechanisms remain incompletely understood. This study employed an integrated approach combining in vitro fermentation systems, Caco-2 cell assays, in vivo murine colitis models, and molecular docking simulations to elucidate the dual mechanisms of 2′-FL action. In vitro fermentation revealed that 2′-FL selectively enhanced propionate and butyrate production while enriching Bacteroides acidifaciens and Allobaculum stercoricanis , key short-chain fatty acids (SCFAs)-producing taxa. In enterotoxigenic Escherichia coli (ETEC)-infected Caco-2 cells, 2′-FL suppressed TLR4/p38 MAPK signaling and inflammation, an effect mirrored by propionate and butyrate. Similarly, in the dextran sulfate sodium (DSS)-induced mouse colitis model, 2′-FL inhibited this signaling pathway, alleviated inflammation, and, in parallel, specifically elevated levels of propionate and butyrate. Molecular docking analyses also indicated that 2′-FL and its metabolites, propionate and butyrate, exhibit binding to the TLR4–MD2 complex with specific affinity. These results support a coordinated mechanism whereby 2′-FL alleviates intestinal inflammation through putative TLR4 antagonism and the microbiota-derived SCFAs, both of which converge to modulate the TLR4/p38 MAPK-dependent signaling axis. Our work provides a mechanistic foundation for 2′-FL as a precision prebiotic targeting gut inflammation via host-microbe crosstalk.