Erythropoietin Protects Intestinal Epithelial Barrier Function and Lowers the Incidence of Experimental Neonatal Necrotizing Enterocolitis

Sheng-Ru Shiou,Yueyue Yu,Sangzi Chen,Mae J Ciancio,Elaine O Petrof,Jun Sun,Erika C Claud

doi:10.1074/jbc.m110.154625

Abstract

The impermeant nature of the intestinal barrier is maintained by tight junctions (TJs) formed between adjacent intestinal epithelial cells. Disruption of TJs and loss of barrier function are associated with a number of gastrointestinal diseases, including neonatal necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal diseases in preterm infants. Human milk is protective against NEC, and the human milk factor erythropoietin (Epo) has been shown to protect endothelial cell-cell and blood-brain barriers. We hypothesized that Epo may also protect intestinal epithelial barriers, thereby lowering the incidence of NEC. Our data demonstrate that Epo protects enterocyte barrier function by supporting expression of the TJ protein ZO-1. As immaturity is a key factor in NEC, Epo regulation of ZO-1 in the human fetal immature H4 intestinal epithelial cell line was examined and demonstrated Epo-stimulated ZO-1 expression in a dose-dependent manner through the PI3K/Akt pathway. In a rat NEC model, oral administration of Epo lowered the incidence of NEC from 45 to 23% with statistical significance. In addition, Epo treatment protected intestinal barrier function and prevented loss of ZO-1 at the TJs in vivo. These effects were associated with elevated Akt phosphorylation in the intestine. This study reveals a novel role of Epo in the regulation of intestinal epithelial TJs and barrier function and suggests the possible use of enteral Epo as a therapeutic agent for gut diseases.

Highlights

It is believed that Zonula occludens-1 (ZO-1) mediates the effect of IFN-␥ on barrier disruption, it is still unclear whether loss of ZO-1 alone is sufficient to disrupt tight junctions (TJs) integrity
In vitro barrier function determined by a FITC-dextran flux experiment on day 16 when control siRNA-transfected cells reached high transepithelial electrical resistance (TER) shows impaired barrier function/increased permeability in ZO-1 knockdown cells compared with controls (Fig. 1C)
These results demonstrate that ZO-1 down-regulation significantly impairs TER and barrier function and that the IFN-␥ effect on barrier disruption can be mediated in part through ZO-1 inhibition in T84 cells

Summary

Introduction

Tight junctions are composed of trans-membrane proteins, including occludin, claudins, and junctional adhesion molecules, as well as cytoplasmic proteins such as zonula occludens (ZO-1, ZO-2, and ZO-3) [4]. These proteins work in concert to form physical connections between epithelial cells and confer basic barrier properties. Previous studies have shown that IFN-␥ treatment impairs barrier function and decreases the TJ protein ZO-1 expression in polarized human T84 enterocytes [10] This suggests that intestinal integrity could be compromised in disease states through altered TJ protein expression. Human milk is protective against NEC as human milk-fed preterm infants are less susceptible to NEC compared with for-

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Results

Conclusion