Lactoferrin and Osteopontin Cooperatively Promote Intestinal Epithelial Maturation in Neonatal Mice by Activating the Brg1/Notch1/Hes1 Pathway

<b>Background/Objectives:</b> Early life is crucial for infant gut development and intestinal homeostasis. Lactoferrin (LF) and osteopontin (OPN) are bioactive breast milk proteins that are supplemented into infant formula to promote gut development. However, the combined effect of LF and OPN (LOP) on in vivo gut maturation has not been fully elucidated. This study investigated the effects of LF, OPN, and LOP on intestinal epithelium maturation in C57BL/6N mice from postnatal days 7 to 21. <b>Methods:</b> 3-day-old pups were assigned to four groups: Control group, LF group: 300 mg/kg LF; OPN group: 300 mg/kg OPN, LOP group: 300 mg/kg of a 1:5 (w/w) mixture of LF and OPN. <b>Results:</b> Compared to controls, LOP reduced plasma Diamine Oxidase (DAO) activity by 1.54-fold and D-lactate levels by 1.41-fold, demonstrating greater efficacy than LF or OPN alone in reducing intestinal permeability. LOP also significantly increased intestinal absorptive cells versus controls or single proteins. Mechanistically, LOP promoted directional intestinal stem cell differentiation, increasing jejunal transit-amplifying cells by 1.40-fold in 21-day-old mice. LOP upregulated expression of the Notch pathway target Hes1 by 1.70-fold. Further investigations revealed LOP activated Notch signaling via the transcription factor Brg1. Validation using intestinal organoids and IEC-6 cells confirmed intact OPN within LOP mediates increased Brg1 expression, activating the Notch pathway to direct intestinal stem cell differentiation into absorptive cells. <b>Conclusions:</b> Collectively, these findings in neonatal mice suggest that LOP cooperatively enhances intestinal barrier maturation and directs stem cell differentiation via Brg1-Notch signaling, offering potential insights for future research on bioactive protein supplementation in infant nutrition.