Maternal Milk 3′-Sialyllactose Promotes Neonatal Intestinal Maturation and Prevents Hyperbilirubinemia through an HNF4α-Associated Pathway

BACKGROUND AND AIMS: Proper development of the neonatal small intestine is critical for nutrient absorption, barrier function, and bilirubin clearance. Maternal milk contains bioactive oligosaccharides such as 3'-sialyllactose (3'SL), while the sialyltransferase St3gal4 regulates endogenous glycosylation. Their contributions to intestinal development and neonatal hyperbilirubinemia, however, remain poorly understood. METHODS: We employed the humanized UGT1 (hUGT1) mouse model, which spontaneously develops neonatal hyperbilirubinemia due to delayed induction of liver and intestinal UGT1A1. By combining this jaundiced background with maternal milk lacking 3'SL, we examined the roles of maternal milk 3'SL and the pup St3gal4 gene in neonatal intestinal development, bilirubin metabolism, and long-term gut function. RESULTS: Neonates deprived of maternal milk 3'SL exhibited reduced survival, growth retardation, shortened intestinal length, and aggravated hyperbilirubinemia, associated with markedly decreased intestinal UGT1A1 expression. Transcriptomic analyses revealed downregulation of intestinal maturation markers and secretory lineage genes, accompanied by upregulation of fetal and pancreatic gene programs. RT-qPCR, immunohistochemistry, and Western blot confirmed reduced expression of goblet cell (Muc2), Paneth cell (Lyz1, Defa5), and UGT1A1 proteins in 3'SL-deficient pups. Mechanistically, St3gal4 deficiency was associated with aberrant activation of pancreatic gene programs, while 3'SL deficiency correlated with reduced HNF4α signaling, together implicating disrupted intestinal epithelial identity. Remarkably, these developmental defects were associated with long-term consequences: St3gal4-deficient mice exhibited heightened susceptibility to DSS-induced colonic injury in adulthood, with greater weight loss and reduced survival. CONCLUSIONS: Collectively, these findings indicate that maternal milk-derived 3'SL and the St3gal4 gene are essential for establishing neonatal intestinal identity, promoting secretory lineage differentiation, and sustaining intestinal UGT1A1 expression. These findings reveal complementary roles of maternal milk-derived 3'SL and St3gal4-dependent sialylation in safeguarding neonatal survival and shaping long-term intestinal resilience.