Human milk fortification increases bnip3 expression associated with intestinal cell death in vitro

Objectives: The aim of the present study was to determine the in vitro effect(s) of a bovine-based human breast milk fortifier (HMF) on human intestinal cells. HMF increases the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein (Bnip3) and cell death; the prostaglandin analogue misoprostol will rescue this effect. Methods: Cultured intestinal cells were exposed to in vitro-digested human breast milk (BM)+HMF. Intracellular oxidation, cell damage/cell death, and BNIP3 expression were measured after exposure. Results: In vitro-digested BMpHMF significantly increased intracellular oxidation, cell damage, and cell death in enterocyte cell cultures compared with either saline or BM controls, an effect that was rescued by the prostaglandin analogue, misoprostol. Bnip3 transcript and Bnip3 protein levels were significantly increased in vitro after treatment with BMpHMF. We also provide evidence that transfection of enterocytes with Bnip3 increases cell death, an effect that is rescued by a nonfunctional Bnip3 splice variant. Conclusions: Our data support the hypothesis that HMF increases intestinal Bnip3 in vitro, and that the gene product triggers cell death. We suggest that misoprostol is a promising therapy, which may reduce intestinal cell death.