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

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. 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. IntroductionIntestinal impermeability is regulated by tight junctions (TJs) 2The abbreviations used are: TJ, tight junction; NEC, neonatal necrotizing enterocolitis; Epo, erythropoietin; IEC, intestinal epithelial cells; ANOVA, analysis of variance; TER, transepithelial electrical resistance. formed between intestinal epithelial cells (IEC) at the most apical areas of the epithelium. Tight junctions serve to establish size- and charge-selective barriers between the gut lumen and the mucosa to control the diffusion of solutes, water, and electrolytes while also serving a key role in host defense to confine toxins, allergens, and pathogens to the intestinal lumen. Disruption of TJs is associated with a number of gastrointestinal diseases, including neonatal necrotizing enterocolitis (NEC). NEC is the leading cause of death from gastrointestinal diseases in premature newborns and is characterized by loss of intestinal barrier function (1Kosloske A.M. Acta Paediatr. Suppl. 1994; 396: 2-7Crossref PubMed Scopus (82) Google Scholar, 2Holman R.C. Stehr-Green J.K. Zelasky M.T. Am. J. Public Health. 1989; 79: 987-989Crossref PubMed Scopus (78) Google Scholar, 3Anand R.J. Leaphart C.L. Mollen K.P. Hackam D.J. Shock. 2007; 27: 124-133Crossref PubMed Scopus (170) Google Scholar). It has been suggested that impaired intestinal barrier may predispose preterm infants to luminal bacteria invasion and immune system activation in the pathogenesis of NEC.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) (4Anderson J.M. Balda M.S. Fanning A.S. Curr. Opin. Cell Biol. 1993; 5: 772-778Crossref PubMed Scopus (181) Google Scholar). These proteins work in concert to form physical connections between epithelial cells and confer basic barrier properties. Zonula occludens-1 (ZO-1), the first TJ protein identified, contains protein-binding domains for interaction with other tight junction-associated proteins, including ZO-2, ZO-3, occludin, β-catenin, paxillin, and talin as well as with the peri-junctional actin ring (5Stevenson B.R. Siliciano J.D. Mooseker M.S. Goodenough D.A. J. Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1272) Google Scholar, 6Schneeberger E.E. Lynch R.D. Am. J. Physiol. Cell Physiol. 2004; 286: C1213-C1228Crossref PubMed Scopus (1097) Google Scholar). Therefore, ZO-1 serves as an important linker between the TJ and the actin cytoskeleton and is thought to be a functionally critical tight junction component.Proinflammatory cytokines are frequently found elevated in intestinal diseases and NEC (7Ford H. Watkins S. Reblock K. Rowe M. J. Pediatr. Surg. 1997; 32: 275-282Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 8Viscardi R.M. Lyon N.H. Sun C.C. Hebel J.R. Hasday J.D. Pediatr. Pathol. Lab. Med. 1997; 17: 547-559Crossref PubMed Scopus (93) Google Scholar). In addition to immune activation, accumulating evidence indicates that proinflammatory cytokines can induce intestinal TJ barrier disruption. IFN-γ is one of the proinflammatory cytokines found elevated in NEC (7Ford H. Watkins S. Reblock K. Rowe M. J. Pediatr. Surg. 1997; 32: 275-282Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 8Viscardi R.M. Lyon N.H. Sun C.C. Hebel J.R. Hasday J.D. Pediatr. Pathol. Lab. Med. 1997; 17: 547-559Crossref PubMed Scopus (93) Google Scholar), and its role in the alteration of the intestinal TJ barrier has been well established (9Madara J.L. Stafford J. J. Clin. Invest. 1989; 83: 724-727Crossref PubMed Scopus (640) Google Scholar, 10Youakim A. Ahdieh M. Am. J. Physiol. 1999; 276: G1279-G1288PubMed Google Scholar, 11Yang H. Kiristioglu I. Fan Y. Forbush B. Bishop D.K. Antony P.A. Zhou H. Teitelbaum D.H. Ann. Surg. 2002; 236: 226-234Crossref PubMed Scopus (70) Google Scholar). Previous studies have shown that IFN-γ treatment impairs barrier function and decreases the TJ protein ZO-1 expression in polarized human T84 enterocytes (10Youakim A. Ahdieh M. Am. J. Physiol. 1999; 276: G1279-G1288PubMed Google Scholar). This suggests that intestinal integrity could be compromised in disease states through altered TJ protein expression. In fact, in animal models of NEC, those with disease had both impaired intestinal barrier function and aberrant TJ protein expression, and in NEC infants, the significantly increased intestinal permeability is also associated with altered TJ protein expression (12Clark J.A. Doelle S.M. Halpern M.D. Saunders T.A. Holubec H. Dvorak K. Boitano S.A. Dvorak B. Am. J. Physiol. Gastrointest. Liver Physiol. 2006; 291: G938-G949Crossref PubMed Scopus (212) Google Scholar). Thus, agents that can reverse the adverse effects of proinflammatory cytokines on TJ proteins and barrier function are expected to minimize barrier disruption following pathological insults in gastrointestinal diseases.Human milk is protective against NEC as human milk-fed preterm infants are less susceptible to NEC compared with formula-fed infants (13Lucas A. Cole T.J. Lancet. 1990; 336: 1519-1523Abstract PubMed Scopus (1225) Google Scholar). Erythropoietin (Epo) is a human milk factor (14Kling P.J. Sullivan T.M. Roberts R.A. Philipps A.F. Koldovský O. Pediatr. Res. 1998; 43: 216-221Crossref PubMed Scopus (83) Google Scholar). Although first described as a major regulator of erythropoiesis, Epo exhibits additional biological activities, including protection of endothelial cell-cell and blood-brain barriers (15Martínez-Estrada O.M. Rodríguez-Millán E. González-De Vicente E. Reina M. Vilaró S. Fabre M. Eur. J. Neurosci. 2003; 18: 2538-2544Crossref PubMed Scopus (147) Google Scholar, 16Li Y. Lu Z.Y. Ogle M. Wei L. Neurochem. Res. 2007; 32: 2132-2141Crossref PubMed Scopus (62) Google Scholar). Functional Epo receptors are present on the luminal side of villi in fetal and neonatal human and rat intestines (17Juul S.E. Yachnis A.T. Christensen R.D. Early Hum. Dev. 1998; 52: 235-249Crossref PubMed Scopus (272) Google Scholar, 18Juul S.E. Joyce A.E. Zhao Y. Ledbetter D.J. Pediatr. Res. 1999; 46: 263-268Crossref PubMed Scopus (124) Google Scholar), suggesting physiological roles of Epo in the developing gut. These findings and previous studies showing the importance of an intact intestinal barrier in limiting the progression of gut diseases collectively lead to our hypothesis that Epo may protect intestinal epithelial barrier function, thereby lowering the incidence of NEC.Our in vitro studies demonstrate that Epo is able to reverse the effect of IFN-γ and protect both TJ protein ZO-1 expression and barrier function. In this study, we also report the protective effects of Epo and reduced NEC incidence in vivo in the immature intestine of an animal model of NEC. The underlying mechanism appears to be mediated through PI3K-dependent activation of Akt. These Epo effects via the PI3K/Akt pathway appear to be specific as oral administration of another human milk factor, transforming growth factor-β (TGF-β), neither activated Akt nor protected intestinal barrier function. This study demonstrates a novel biological function of Epo and suggests a potential use for Epo in gut diseases.DISCUSSIONNEC is the most common cause of death from gastrointestinal diseases in preterm infants. Previous studies have revealed the protective effects of breast milk on both the incidence and the severity of NEC (36Kurscheid T. Holschneider A.M. Eur. J. Pediatr. Surg. 1993; 3: 139-143Crossref PubMed Scopus (36) Google Scholar). Components in human milk responsible for this protective effect are expected to act on steps in the pathogenesis of NEC. Understanding the underlying mechanisms of biochemical cross-talk between human milk factors and the intestines of breastfed infants offers an opportunity for development of therapeutic agents for this disease.Erythropoietin is found in human milk. Although first reported as a major stimulator of erythropoiesis in erythroid progenitor cells, several studies have indicated diverse effects of Epo in nonhematopoietic cells with Epo receptors, including cardiac muscle cells and neurons, where Epo acts as a protective cytokine (37Madonna R. Shelat H. Xue Q. Willerson J.T. De Caterina R. Geng Y.J. Exp. Cell Res. 2009; 315: 2921-2928Crossref PubMed Scopus (16) Google Scholar, 38Liu R. Suzuki A. Guo Z. Mizuno Y. Urabe T. J. Neurochem. 2006; 96: 1101-1110Crossref PubMed Scopus (84) Google Scholar, 39Yoo J.Y. Won Y.J. Lee J.H. Kim J.U. Sung I.Y. Hwang S.J. Kim M.J. Hong H.N. J. Neurosci. Res. 2009; 87: 150-163Crossref PubMed Scopus (30) Google Scholar). Functional Epo receptors are also present at the IEC brush border of fetal and postnatal intestines in humans and rats, suggesting a role of Epo in the developing gut (18Juul S.E. Joyce A.E. Zhao Y. Ledbetter D.J. Pediatr. Res. 1999; 46: 263-268Crossref PubMed Scopus (124) Google Scholar). Our data demonstrate that Epo can specifically preserve intestinal barrier function under conditions of inflammatory stress. Our results thus reveal a novel protective effect of Epo on intestinal barrier integrity.We found that Epo protected enterocyte barrier function via maintenance of ZO-1 levels and cellular localization in vitro in the cell line H4. As the immature H4 cells do not polarize or form sufficient tight junctions to measure barrier function, we measured the effect of Epo on ZO-1 and barrier function on immature intestinal epithelia under conditions of inflammatory stress in vivo. Our data in newborn rat pups demonstrated that Epo significantly decreased NEC incidence, attenuated intestinal barrier disruption, and maintained TJ structure and ZO-1 localization in vivo in an experimental NEC animal model.NEC is known to be associated with overexpression of a number of proinflammatory cytokines. In addition to inducing an inflammatory response, proinflammatory cytokines are now known to induce barrier disruption. IFN-γ has been shown to be elevated in NEC (7Ford H. Watkins S. Reblock K. Rowe M. J. Pediatr. Surg. 1997; 32: 275-282Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 40Caplan M.S. Sun X.M. Hseuh W. Hageman J.R. J. Pediatr. 1990; 116: 960-964Abstract Full Text PDF PubMed Scopus (268) Google Scholar) and a mediator for inducing intestinal epithelial permeability both in vitro and in vivo (10Youakim A. Ahdieh M. Am. J. Physiol. 1999; 276: G1279-G1288PubMed Google Scholar, 11Yang H. Kiristioglu I. Fan Y. Forbush B. Bishop D.K. Antony P.A. Zhou H. Teitelbaum D.H. Ann. Surg. 2002; 236: 226-234Crossref PubMed Scopus (70) Google Scholar, 41Ferrier L. Mazelin L. Cenac N. Desreumaux P. Janin A. Emilie D. Colombel J.F. Garcia-Villar R. Fioramonti J. Bueno L. Gastroenterology. 2003; 125: 795-804Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Correspondingly, NEC has been shown to involve structural and functional loss of the intestinal barrier as described in the study by Piena-Spoel et al. (42Piena-Spoel M. Albers M.J. ten Kate J. Tibboel D. J. Pediatr. Surg. 2001; 36: 587-592Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), in which human neonates diagnosed with severe cases of NEC had increased intestinal permeability compared with healthy controls. From our study, loss of the TJ protein ZO-1 important for barrier function was in to NEC stress that not have evidence of NEC. This suggests that the ZO-1 loss we was not a of associated with intestinal of the disease could be an that intestinal barrier function in pathological conditions has been associated with altered expression of TJ as well as junctional we found elevated and in the experimental NEC compared with are other TJ an important role in the regulation of S. M. Ann. PubMed Scopus Google Scholar). expression was between experimental NEC and healthy In expression was in experimental NEC intestines where increased intestinal permeability was have shown that can be used as a of intestinal J. A. E. J. Clin. PubMed Scopus Google Scholar). Our findings are with the study by et al. (12Clark J.A. Doelle S.M. Halpern M.D. Saunders T.A. Holubec H. Dvorak K. Boitano S.A. Dvorak B. Am. J. Physiol. Gastrointest. Liver Physiol. 2006; 291: G938-G949Crossref PubMed Scopus (212) Google Scholar) in which increased was in NEC intestines and with the of NEC It is possible that intestinal epithelial cells expression of important for barrier function to for the barrier disruption as the intestine of expression of in the in with disease also demonstrated the between elevated and increased intestinal permeability H. A. A. K. M. A. PubMed Scopus Google Scholar). The are thought to be the proteins that the of the TJ barrier S. M. J. Cell Biol. PubMed Scopus Google Scholar) and the increased expression may a for increased intestinal Epo administration ZO-1 not significantly expression of or in experimental NEC.Our in vivo data Akt activation in experimental NEC which can be increased by Epo treatment to healthy intestinal control suggesting a role of Akt activation in Epo of ZO-1 and intestinal barrier function in vivo. The Akt pathway has been shown to be in the regulation of barrier integrity in endothelial cells. endothelial barrier integrity by inducing ZO-1 to the cell-cell junctional J.F. Q. H. L. T. E. Lee M.J. J. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), and the effect of was of PI3K/Akt The Akt pathway is one of the known of Epo and was activated in the immature H4 In cells, activation mediated Epo regulation of ZO-1 as with and the for the Akt Epo of ZO-1. Epo of ZO-1 expression was also significantly in the of the and suggesting that the and are also in regulation of ZO-1 expression. Epo Akt not and phosphorylation as by against and It is possible that the and activation may be for regulation of ZO-1 expression and that Akt activation via Epo regulation of ZO-1 in immature is the first study to the effects of enteral Epo on IEC and experimental NEC. have the effect of Epo via study of the of Epo administration for and treatment of of demonstrated lowered incidence of NEC in infants D.J. S.E. J. Pediatr. Surg. Full Text Full Text PDF PubMed Scopus Google Scholar). In animal of human Epo in a rat model of NEC to stress associated with the pathogenesis of NEC M. M. A. N. Eur. J. Pediatr. Surg. 2001; PubMed Scopus Google Scholar, A. H. N. O. M. E. N. S. H. Biol. 2003; PubMed Scopus Google Scholar). of through disruption, and as have Epo receptors, administration a for Our study used immature rat pups by and oral administration of rat Epo to specifically Epo has effects on IEC and Epo can protect an immature intestine from NEC through its intestinal In our model, NEC is a and can be thus our study on of disease This is with Epo as a of protective human milk. Our study demonstrates that Epo is able to its against the of NEC the gut following oral administration at physiological Epo was used at a the physiological of breast milk to the protective role of breast milk and to specifically demonstrate that protection could be at this data that enteral administration of Epo at with those found in human milk is sufficient for maintenance of ZO-1 expression in the of inflammatory leading to of barrier function and reduced incidence of NEC in an animal As Epo is an and agent and has been used to in infants, this study not an important first the of an factor in breast milk in infants from NEC also suggests for the novel therapeutic use of Epo in intestinal diseases. IntroductionIntestinal impermeability is regulated by tight junctions (TJs) 2The abbreviations used are: TJ, tight junction; NEC, neonatal necrotizing enterocolitis; Epo, erythropoietin; IEC, intestinal epithelial cells; ANOVA, analysis of variance; TER, transepithelial electrical resistance. formed between intestinal epithelial cells (IEC) at the most apical areas of the epithelium. Tight junctions serve to establish size- and charge-selective barriers between the gut lumen and the mucosa to control the diffusion of solutes, water, and electrolytes while also serving a key role in host defense to confine toxins, allergens, and pathogens to the intestinal lumen. Disruption of TJs is associated with a number of gastrointestinal diseases, including neonatal necrotizing enterocolitis (NEC). NEC is the leading cause of death from gastrointestinal diseases in premature newborns and is characterized by loss of intestinal barrier function (1Kosloske A.M. Acta Paediatr. Suppl. 1994; 396: 2-7Crossref PubMed Scopus (82) Google Scholar, 2Holman R.C. Stehr-Green J.K. Zelasky M.T. Am. J. Public Health. 1989; 79: 987-989Crossref PubMed Scopus (78) Google Scholar, 3Anand R.J. Leaphart C.L. Mollen K.P. Hackam D.J. Shock. 2007; 27: 124-133Crossref PubMed Scopus (170) Google Scholar). It has been suggested that impaired intestinal barrier may predispose preterm infants to luminal bacteria invasion and immune system activation in the pathogenesis of NEC.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) (4Anderson J.M. Balda M.S. Fanning A.S. Curr. Opin. Cell Biol. 1993; 5: 772-778Crossref PubMed Scopus (181) Google Scholar). These proteins work in concert to form physical connections between epithelial cells and confer basic barrier properties. Zonula occludens-1 (ZO-1), the first TJ protein identified, contains protein-binding domains for interaction with other tight junction-associated proteins, including ZO-2, ZO-3, occludin, β-catenin, paxillin, and talin as well as with the peri-junctional actin ring (5Stevenson B.R. Siliciano J.D. Mooseker M.S. Goodenough D.A. J. Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1272) Google Scholar, 6Schneeberger E.E. Lynch R.D. Am. J. Physiol. Cell Physiol. 2004; 286: C1213-C1228Crossref PubMed Scopus (1097) Google Scholar). Therefore, ZO-1 serves as an important linker between the TJ and the actin cytoskeleton and is thought to be a functionally critical tight junction component.Proinflammatory cytokines are frequently found elevated in intestinal diseases and NEC (7Ford H. Watkins S. Reblock K. Rowe M. J. Pediatr. Surg. 1997; 32: 275-282Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 8Viscardi R.M. Lyon N.H. Sun C.C. Hebel J.R. Hasday J.D. Pediatr. Pathol. Lab. Med. 1997; 17: 547-559Crossref PubMed Scopus (93) Google Scholar). In addition to immune activation, accumulating evidence indicates that proinflammatory cytokines can induce intestinal TJ barrier disruption. IFN-γ is one of the proinflammatory cytokines found elevated in NEC (7Ford H. Watkins S. Reblock K. Rowe M. J. Pediatr. Surg. 1997; 32: 275-282Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 8Viscardi R.M. Lyon N.H. Sun C.C. Hebel J.R. Hasday J.D. Pediatr. Pathol. Lab. Med. 1997; 17: 547-559Crossref PubMed Scopus (93) Google Scholar), and its role in the alteration of the intestinal TJ barrier has been well established (9Madara J.L. Stafford J. J. Clin. Invest. 1989; 83: 724-727Crossref PubMed Scopus (640) Google Scholar, 10Youakim A. Ahdieh M. Am. J. Physiol. 1999; 276: G1279-G1288PubMed Google Scholar, 11Yang H. Kiristioglu I. Fan Y. Forbush B. Bishop D.K. Antony P.A. Zhou H. Teitelbaum D.H. Ann. Surg. 2002; 236: 226-234Crossref PubMed Scopus (70) Google Scholar). Previous studies have shown that IFN-γ treatment impairs barrier function and decreases the TJ protein ZO-1 expression in polarized human T84 enterocytes (10Youakim A. Ahdieh M. Am. J. Physiol. 1999; 276: G1279-G1288PubMed Google Scholar). This suggests that intestinal integrity could be compromised in disease states through altered TJ protein expression. In fact, in animal models of NEC, those with disease had both impaired intestinal barrier function and aberrant TJ protein expression, and in NEC infants, the significantly increased intestinal permeability is also associated with altered TJ protein expression (12Clark J.A. Doelle S.M. Halpern M.D. Saunders T.A. Holubec H. Dvorak K. Boitano S.A. Dvorak B. Am. J. Physiol. Gastrointest. Liver Physiol. 2006; 291: G938-G949Crossref PubMed Scopus (212) Google Scholar). Thus, agents that can reverse the adverse effects of proinflammatory cytokines on TJ proteins and barrier function are expected to minimize barrier disruption following pathological insults in gastrointestinal diseases.Human milk is protective against NEC as human milk-fed preterm infants are less susceptible to NEC compared with formula-fed infants (13Lucas A. Cole T.J. Lancet. 1990; 336: 1519-1523Abstract PubMed Scopus (1225) Google Scholar). Erythropoietin (Epo) is a human milk factor (14Kling P.J. Sullivan T.M. Roberts R.A. Philipps A.F. Koldovský O. Pediatr. Res. 1998; 43: 216-221Crossref PubMed Scopus (83) Google Scholar). Although first described as a major regulator of erythropoiesis, Epo exhibits additional biological activities, including protection of endothelial cell-cell and blood-brain barriers (15Martínez-Estrada O.M. Rodríguez-Millán E. González-De Vicente E. Reina M. Vilaró S. Fabre M. Eur. J. Neurosci. 2003; 18: 2538-2544Crossref PubMed Scopus (147) Google Scholar, 16Li Y. Lu Z.Y. Ogle M. Wei L. Neurochem. Res. 2007; 32: 2132-2141Crossref PubMed Scopus (62) Google Scholar). Functional Epo receptors are present on the luminal side of villi in fetal and neonatal human and rat intestines (17Juul S.E. Yachnis A.T. Christensen R.D. Early Hum. Dev. 1998; 52: 235-249Crossref PubMed Scopus (272) Google Scholar, 18Juul S.E. Joyce A.E. Zhao Y. Ledbetter D.J. Pediatr. Res. 1999; 46: 263-268Crossref PubMed Scopus (124) Google Scholar), suggesting physiological roles of Epo in the developing gut. These findings and previous studies showing the importance of an intact intestinal barrier in limiting the progression of gut diseases collectively lead to our hypothesis that Epo may protect intestinal epithelial barrier function, thereby lowering the incidence of NEC.Our in vitro studies demonstrate that Epo is able to reverse the effect of IFN-γ and protect both TJ protein ZO-1 expression and barrier function. In this study, we also report the protective effects of Epo and reduced NEC incidence in vivo in the immature intestine of an animal model of NEC. The underlying mechanism appears to be mediated through PI3K-dependent activation of Akt. These Epo effects via the PI3K/Akt pathway appear to be specific as oral administration of another human milk factor, transforming growth factor-β (TGF-β), neither activated Akt nor protected intestinal barrier function. This study demonstrates a novel biological function of Epo and suggests a potential use for Epo in gut diseases.

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