Human milk-derived disialyllacto-N-tetraose protects against necrotizing enterocolitis by suppressing PI3K/AKT-mediated macrophage pro-inflammatory activation

Necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality in preterm infants, and human milk oligosaccharides (HMOs) have emerged as bioactive components implicated in its prevention. Although reduced disialyllacto-N-tetraose (DSLNT) in maternal milk has been linked to NEC risk, the molecular mechanism by which DSLNT modulates intestinal inflammation remains poorly defined. UPLC-MS/MS quantification of ten representative HMOs in milk from mothers of full-term, preterm, and NEC infants revealed a reduction of DSLNT in the NEC group. An integrated strategy combining network pharmacology, KEGG enrichment analysis, molecular docking, and molecular dynamics simulations predicted the PI3K/AKT signaling pathway as a potential target of DSLNT. Functional validation in LPS/IFN-γ-stimulated RAW 264.7 macrophages demonstrated that DSLNT inhibited pro-inflammatory activation, alleviated oxidative stress, stabilized mitochondrial function, and suppressed iNOS expression as well as AKT phosphorylation; these protective effects were abrogated by the PI3K agonist 740Y-P. Mechanistic dissection further confirmed that DSLNT suppresses the activation of the PI3K/AKT pathway, thereby downregulating the expression of inflammatory mediators. Collectively, these findings support that DSLNT attenuates the inflammatory response through inhibition of the PI3K/AKT pathway in macrophages, providing a mechanistic rationale for its further evaluation as a therapeutic candidate.