Human Milk Oligosaccharides Activate Epidermal Growth Factor Receptor and Protect Against Hypoxia-Induced Injuries in the Mouse Intestinal Epithelium and Caco2 Cells

ABSTRACT

Background:

Hypoxia-induced intestinal barrier injuries lead to necrotizing enterocolitis (NEC). Although NEC in preterm neonates is preventable by human milk oligosaccharides (HMOs), the underlying mechanism remains unknown.

Objective:

To reveal the role and mechanism of HMOs in protecting against hypoxia-induced injuries in intestinal epithelium of neonatal mice and cultured Caco2 cells.

Methods:

NEC was induced by hypoxia and cold stress. Seventy C57BL/C pups (7-d-old) were divided into 5 groups and fed maternal breast milk (BM), formula alone (FF), or the formula added with HMOs at 5 (LHMO), 10 (MHMO), or 20 mg/mL (HHMO) for 3 d. Ileal hypoxia inducible factor 1a (HIF1a) and cleaved Caspase 3 were determined, along with staining for Ki-67 protein to labeled proliferative cells. In vitro, adherent Caco2 cells (undifferentiated, passage 14) were treated with HMOs, galacto-oligosaccharides, fructo-oligosaccharides, or mixed oligosaccharides at 10 mg/mL for 1 d exposed to 1% O₂. Cell proliferation and apoptosis, along with phosphorylated epidermal growth factor receptor (P-EGFR) and 38KD MAPK (P-P38), were assayed in differentiated or undifferentiated Caco2 cells.

Results:

Compared with the FF-fed mice, those fed MHMO and HHMO had 52% lower (P< 0.05) NEC scores, 60–80% greater (P< 0.05) KI67-positive cell numbers, and 56–71% decreases (P< 0.05) in ileal HIF1a and cleaved Caspase 3 (56–71%). Compared with those untreated, the HMO-treated Caco2 cells displayed 60% greater (P< 0.05) proliferative activity and 19% lower (P< 0.05) apoptotic cells after the hypoxia exposure. The HMO treatment led to 58% or 10-fold increases (P< 0.05) of P-EGFR and 48–89% decreases (P< 0.05) of P-P38 in either differentiated or undifferentiated Caco2 cells compared with the controls.

Conclusion:

Supplementing HMOs at 10–20 mg/mL into the formula for neonatal mice or media for Caco2 cells conferred protection against the hypoxia-induced injuries. The protection in the Caco2 cells was associated with an activation of EGFR.