| IntroductionNecrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in the newborn infants, occurring primarily in low birthweight premature infants. The overall incidence of NEC is about1per1000live births, but it occurs in up to7%of very low birthweight infants. However, it is likely to continue to rise because advances in neonatology and the modern NICU over the last few decades have increased survival of very low birth weight infants, which results in a greater number of neonates at risk of developing NEC. Despite advances in neonatal intensive care, the overall mortality from NEC remains high, ranging from15%to30%, and survivors often experience severe long-term complications including intestinal strictures, short bowel syndrome, recurrent sepsis, poor growth and neurodevelopmental impairment. In severely affected infants, characterized by bowel wall necrosis, perforation, peritonitis, bacterial invasion, systemic sepsis and multi-system organ failure, the mortality even approaches100%. Although prematurity, formula feeding, intestinal hypoxia-ischemia, inappropriate bacterial colonization are considered major risk factors of NEC, the precise etiology of NEC remains elusive and no effective treatments are currently available. Recently, studies have focused on the role of the inflammatory cascade and its impact on the disease process. Here NEC was suggested as an uncontrolled exuberant inflammatory response to bacterial colonization that characterizes the intestine of premature infant. Inflammation was considered to play a central role in its pathogenesis. Although many inflammatory mediators,receptors and signaling pathways are involved in the pathophysiology process, the key role of inflammation in the development of NEC and as a potential target for preventive or therapeutic measures remain unknown.TLRs are a family of pattern recognition receptors (PRRs) that play a critical role in the activation of the innate immune system in response to microbial invaders, widely expressed on the surface of a variety of host defense cells, recognizing specific pathogen regions or molecular motifs, referred to as pathogen-associated molecular patterns (PAMPs). Among the TLRs,TLR4was the first identified and most thoroughly studied member, which is the receptor for lipopolysaccharides (LPS), the outer membrane component of Gram-negative bacteria. Recent studies have shed light on the important and exciting role of the innate immune Toll like receptor4(TLR4) of the intestinal epithelium in the development of NEC. It is now known that human TLR4is expressed at very low levels, but studies in both NEC animal models and resected intestine from infants with necrotizing enterocolitis indicate that NEC is associated with increased expression of TLR4in the intestinal mucosa compared with those infants and animals without NEC.Prematurity,hypoxia,formula feeding,and bacteria—these risk factors for NEC may lead to persistent up-regulation of intestinal TLR4. TLR4activation triggers a cascade of signaling molecules and ultimately activates the transcription factor NF-κB, a key transcriptional regulator of innate and adaptive immunity. When activated, NF-kB translocates into the cell's nucleus and switches on the genes responsible for the expression of inflammatory mediators, inducing the expression of various inflammatory cytokines, an exaggerated inflammatory mediator response all contributing to the development of epithelial cell injury and a weakened mucosal defense system that fosters the development of NEC, and leading to bacterial translocation from the intestinal lumen and leukocyte activation, stimulating local and systemic cytokine release, resulting in a systemic inflammatory response, sepsis and multiorgan failure. Because NEC often progresses from subtle signs to extensive necrosis, intestinal perforation, and peritonitis within hours, prophylactic measures are preferred.Human milk has protective effect against NEC because it contains multiple bioactive substances that can exert anti-inflammatory and antimicrobial effect. One of the many biologically active growth factors present in human and animal milk is erythropoietin,and functional EPO receptors are present on enterocytes of fetal and neonatal small bowel on the luminal side of villi in fetal and neonatal human and rat intestines. The latter suggests that EPO has a physiological role in growth and development of the gastrointestinal tract. Although first described as a major regulator of erythropoiesis, EPO exhibits additional non-erythropoietic biological activities including inhibiting apoptosis and inflammation, and inducting angiogenesis. Interestingly, a retrospective study of preterm infants administrated with EPO via injection(in a daily dose of200U/kg intravenously as a continuous infusion in the hyperalimentation solution or as400U/kg subcutaneously,3days/week) for the prevention and treatment of the anemia showed a decreased incidence of NEC compared to infants who did not receive EPO, suggesting that EPO has a protective effect against NEC. But injection potentially carries risks of infection through skin disruption and as EPO receptor widely expressed in several nonhematopoietic tissues,systemic administration potentially carries a greater risk for systemic effects,whereas enterally administered EPO appears to have limited systemic absorption, but rather acts locally.The aim of this study is to determine whether enteral erythropoietin supplementation at physiological concentration as in human milk (0.1u/ml) protects against experimental NEC and what the possible mechanisms involved in this process are. In a rat model of NEC induced by formula feeding and hypoxia/cold stress, we examined the efficacy of EPO on the dynamic expression of intestinal TLR4and NF-κB mRNA transcripts and IL-6, SIgA, MUC2protein, and evaluates the effect of EPO on bacterial translocation rate and severity and incidence of NEC.Materials and MethodsMaterials1. Animals:Three-day-old specific pathogen-free (SPF) Sprague-Dawley (SD) neonatal rats weighing between5.12g and10.20g (Guangzhou University of Chinese Medicine Laboratory Animal center, Guangzhou, China) were housed in a SPF room in our Laboratory Animal Center. The rats were kept under controlled temperature (28℃～30℃) and humidity (45%～65%) and with12h dark/light cycles.2. Kit and drugs:SMA PDF GOLD preterm infant formula (Wyeth, USA), Rat Mucin2(MUC-2) ELISA Kit (Shanghai BlueGene Biotech CO., LTD), Rat secretory immunoglobulin A (slgA) ELISA Kit (Shanghai BlueGene Biotech CO., LTD), Rat IL-6ELISA Kit (R&D Systems, USA), EPO(SHENYANG SUNSHINE PHARMACEUTICAL CO.,LTD)3. Equipment:BX50light microscope (Olympus, Japan), RM2255paraffin slicing machine (LeiCA, Germany),7500Fluorescence quantitative PCR instrument (ABI, USA),9700PCR instrument (ABI, USA).Methods1. Neonatal rat NEC model The experimental protocol was designed according to Guide for the Care and Use of Laboratory Animals from The Chinese National Institutes of Health and approved by our Animal Care and Use Committee. The animal model we selected is a slightly modified version of a well-validated NEC model that utilizes preterm mouse pups delivered by Cesarean section, gavage formula feeding and hypoxia/cold stress to induce NEC.Three-day-old SD rat pups were separated from their mothers, housed in an incubator, and fed with formula four times daily via orogastric gavage using a24G silastic catheter, then experienced hypoxia (breathing100%nitrogen gas for90s) and cold stress (4℃for10min) three times daily for3days to induce NEC. The milk replacer was configured according to Auestad's method. The feeding volume began at0.15ml and was increased incrementally up to0.35ml.2. Experimental designsExperiment1To investigate the effect of erythropoietin against necrotizing enterocolitis, this study was designed to observe the intestinal histopathological changes and bacterial translocation (BT) in neonatal rat model with NEC. Seventy-five three-day-old SD rat pups were divided randomly into three groups (n=25in each group):normal control group, NEC model group and EPO intervention group.The rat pups in normal control group were kept with their mothers and breast fed, received no other intervention. NEC model group rats were separated from their mothers, housed in an incubator, and gavaged with rat-milk substitute, then experienced hypoxia (breathing100%nitrogen gas for90s) and cold stress (4℃for10min) three times daily for3days. EPO intervention group rats were fed with the substitute of rat-milk supplemented with0.1u/ml of EPO, and also given hypoxia and cold stress similar to the NEC group. Blood samples via cardiac puncture and2-cm lengths of terminal ileum proximal to the ileocecal valve were obtained from the animals on72h. The last2-cm of terminal ileum was fixed in10%neutral formalin, paraffin-embedded, microtome-sectioned at3μm, and stained with hematoxylin and eosin (H&E) for histological evaluation of NEC under a light microscope. Blood samples were stored at-80℃for bacteria DNA extraction.Experiment2To study the effect of erythropoietin on dynamic changes of TLR4mRNA,NF-κB mRNA,IL-6, SIgA and MUC2in necrotizing enterocolitis rat model,we further repeated the same protocol for the three groups,the rat pups were euthanized on24h,48h and72h, respectively (10rats/group). The last3-cm of terminal ileum was excised and averagely cut into halves. One half of each ileum sample was washed with cold phosphate-buffered saline, pH7.4(PBS) and fresh frozen immediately in liquid nitrogen and stored at-80℃for RT-PCR and ELISA assays; the other half was fixed in10%neutral formalin, paraffin-embedded, microtome-sectioned at3μm, and stained with hematoxylin and eosin (H&E) for histological evaluation of NEC under a light microscope.3. Observation of parameters and methods(1) The appearance,weight of rat, death information as well as diarrhoea was recorded daily.(2) The pathology change of the intestine.①Assessment of macroscopic damage.Following incision of the abdomen, the small intestine was evaluated visually for typical gross signs of NEC such as intestinal distension, intestinal wall hemorrhage, or necrosis.②Assessment of microscopic damage under the light microscope. Histological changes in the ileum were scored by two "blinded" independent observers using a previously published NEC scoring system to evaluate the degree of intestinal injury on a "0" to "4" scale as follows:0(normal), no damage;1(mild), slight submucosal and/or lamina propria separation;2(moderate),moderate separation of submucosa and/or lamina propria,and/or edema in submucosal and muscular layers;3(severe), severe separation of submucosa and/or lamina propria, and/or severe edema in submucosa and muscular layers, region villous sloughing;4(necrosis),loss of villi and necrosis. The final score for each animal was determined based upon the highest score observed in any particular specimen. Based on this scoring system, Scores>2are defined as NEC.(3) Bacterial Translocation. To determine the incidence of bacterial translocation, real-time fluorescence quantitative PCR was used to detect the bacterial16S rRNA gene in blood samples.If the Ct value is evident within40PCR cycles,the sample is regarded as positive for bacterial translocation,otherwise the sample is considered as negative.(4) Fluorescence quantitative PCR was applied to evaluate the dynamic levels of TLR4and NF-κB mRNA of the intestinal tissues.(5) The dynamic protein expression of IL-6,SIgA and MUC2were examined by EL1SA methods.4.Statistical AnalysisThe data were analyzed using the SPSS (Statistical Package for Social Sciences)17.0software.Measurement data are expressed as mean values±standard deviations. Comparisons between groups were performed with one-way analysis of variance followed by post hoc tests for multiple comparisons. Differences for the histopathological scores between groups were analyzed by nonparametric Kruskal-Wallis analysis and χ2test was utilized to analyze the incidence of NEC and BT. A P value<0.05was considered statistically significant.ResultsResults of experiment11.Clinical evaluation of rat pups By48-72h of stress, some animals in NEC group appeared lazy, less activity,dispirited, anorexia,diarrhea, abdominal distention and weight loss,etc. The seriously sick rat even developed frequent vomiting, cyanosis, respiratory distress, and pallor et al. Whereas in EPO group, the clinical manifestation lessened compared with NEC group and the body weight increased incrementally. In normal control group, the rat were survived well. Three rats (two in NEC group and one in EPO group) died prior to the72h experimental endpoint, presumably due to mechanical issues associated with feeding (aspiration, esophageal perforation, etc.); these animals were not included in the final analysis. The survival rates for these studies are:normal control group,100%(25/25); NEC model group,92%(23/25); EPO intervention group,96%(24/25).2. Macroscopic observation Macroscopic examination of intestine in NEC group showed hyperaemia, oedema, intestinal distension. The change of intestine in EPO group was lightened compared with NEC group. There was no change in intestine of normal control group.3.The change of intestinal tissues under light microscope In normal control group,intestinal villi was integrity and epithelial cells was lined up in order and no pathological changes was observed. In NEC group, the epithelial cell rank was derangement, inflammatory cell infiltrated, congestion of villus interstitial substance, edema in submucosal and muscular layers, separation of submucosa and/or lamina propria and myometrial thining was apparent with a total loss of villi in some cases. Compared with NEC group, the change of intestinal injury was lightened and the extent of inflammatory cell penetration was reduced in EPO group, and the mean rank of the intestinal histopathological score (39.4583vs53.8696) was significantly lower, P<0.05.4.NEC incidence Histological NEC scores and the incidence of NEC in the experimental groups are plotted individually and summarized according to whether they had a score of2or greater indicative of NEC. The incidence of NEC was markedly decreased to25%(6/24) in the EPO intervention group compared with57%(13/23)in the NEC group (P<0.05). In normal control group,the incidence of NEC was0%(0/25).5. Bacterial Translocation The bacterial translocation rate was significantly reduced to17%(4/24) in the EPO intervention group compared with65%(15/23)in the NEC group (P<0.05). In normal control group,BT rate was0%(0/25).Results of experiment21. Dynamic changes of TLR4,NF-κB mRNA and IL-6protein in different groups at all time points.(1) The TLR4mRNA expression in NEC model group increased at24h, peaked at48h and slightly decreased at72h.Compared with the control group, the expression of all observed time points increased significantly (P<0.01).TLR4mRNA level in EPO group was also found to express highly at24h, with a decline later. The expression at48h and72h was significantly lower than that of model group(P<0.01). There was weak expression of TLR4mRNA in normal control group.(2) The levels of NF-κB mRNA and IL-6in NEC model group were significantly higher than those in control group at48h and72h(P<0.01). The expression of EPO group was observed to increase highly at48h, while the level at72h was significantly lower than that of model group (P<0.01). In normal control group,the expression was steady state with low expression.2. Dynamic changes of MUC2and SIgA protein in different groups at all time points.(1) MUC2expression in ileum of the control group animal was in mean steady-state, while the expression of the NEC experimental group was low in the first24h, rapidly increased to the normal level and reached to the steady-state at48h and72h respectively after the animal model was created. In the EPO intervention group, MUC2expression was found to rapidly increase and reach to high level at48h and72h respectively. Compared with control group, the expression of MUC2in NEC experimental group animal was observed to decrease significantly at24h (P<0.01) without some alterations at48h and72h. Compared with the control group and NEC group, MUC2expression of the EPO intervention group animal was observed to increase significantly (P<0.05).(2) SIgA expression in the control group animal was in average steady-state, while the expression of the NEC experimental group was found to decrease in all time points observed (P<0.01). Compared with the control group, SIgA expression in the EPO group was found to be in low level at24h and48h (P<0.01) without significant change at72h. However, compared with NEC group especially at72h, the SIgA expression was found to significantly increase in EPO intervention group (P<0.01).3.The intestinal histopathological changes No apparent pathological changes were found in all groups at the time points of24h and48h.However, tissue lesion was observed in both model and EPO groups at72h, and score of histological evaluation of model group (2.83±0.56) was significantly higher than that of EPO group (1.53±0.35,P<0.01).Conclusions1.By formula feeding and hypoxia/cold stress, we could induce neonatal rat model which had intestine injury just similar to neonatal necrotizing enterocolitis. Enteral erythropoietin supplementation at physiological concentration as in human milk result in improvement of clinical symptoms and reduction of severity and incidence of NEC.2. In the intestinal tract of the neonatal rat with NEC, innate immune response initiates earlier than acquired immune response,and inflammatory response appears ahead of tissue lesion, which indicates that inflammatory response induces lesion. Enteral administration of EPO at physiologic concentration in human milk may down-regulate TLR4mRNA expression and then inhibit the release of inflammatory mediators, thus reducing the intestinal tissue injury.3. The damaged function of intestinal mucosal barrier in NEC model neonatal rat can be prevented by enteral administration of erythropoietin supplementation at physiological concentration. EPO may protect function of both intestinal mechanical and immune barriers against damage by upregulating the expression of MUC2and SIgA.4. EPO may inhibit the pathogenesis of NEC by decreasing bacterial translocation.
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