Carbon monoxide protects against the development of experimental necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a disease of neonates that is increasing in incidence and often results in significant morbidity and mortality. Carbon monoxide (CO), a byproduct of the catabolism of heme, is known to have anti-inflammatory and antiapoptotic properties. In this study, we aimed to demonstrate that inhaled CO protects against the development of intestinal inflammation in a model of experimental NEC as well as decreases enterocyte cell death in vitro. Additionally, we also aimed to demonstrate that CO decreases enterocyte production of inducible nitric oxide synthase (iNOS) and nitric oxide (NO). Neonatal rats were exposed to intermittent hypoxia exposure and formula feeding to induce experimental NEC. Animals randomized to CO treatment were put in an environment containing 0.025% CO for 1 h/day on days 1-3 of life. All animals were killed on day 4 of life. In vitro experiments were performed with IEC-6 cells, a rat enterocyte cell line. Cells were examined for viability, iNOS production, and elaboration of NO. We found that CO diminished levels of serum inflammatory cytokines and nitrites, protected against intestinal inflammation, and decreased ileal iNOS production and protein nitration in a model of experimental NEC. In vitro, CO decreased cytokine- or hypoxia/endotoxin-induced iNOS and NO production. CO also abrogated TNF-alpha- and actinomycin D-induced apoptosis or hypoxia/endotoxin-induced cell death. In conclusion, 1 h of daily low-dose inhaled CO protected against the development of intestinal inflammation in a model of experimental NEC. iNOS and NO production were decreased by CO both in vivo and in vitro. CO may prove to be a useful clinical adjunct in the treatment of NEC.     

Fecal microbiota in premature infants prior to necrotizing enterocolitis

Intestinal luminal microbiota likely contribute to the etiology of necrotizing enterocolitis (NEC), a common disease in preterm infants. Microbiota development, a cascade of initial colonization events leading to the establishment of a diverse commensal microbiota, can now be studied in preterm infants using powerful molecular tools. Starting with the first stool and continuing until discharge, weekly stool specimens were collected prospectively from infants with gestational ages ≤32 completed weeks or birth weights≤1250 g. High throughput 16S rRNA sequencing was used to compare the diversity of microbiota and the prevalence of specific bacterial signatures in nine NEC infants and in nine matched controls. After removal of short and low quality reads we retained a total of 110,021 sequences. Microbiota composition differed in the matched samples collected 1 week but not <72 hours prior to NEC diagnosis. We detected a bloom (34% increase) of Proteobacteria and a decrease (32%) in Firmicutes in NEC cases between the 1 week and <72 hour samples. No significant change was identified in the controls. At both time points, molecular signatures were identified that were increased in NEC cases. One of the bacterial signatures detected more frequently in NEC cases (p<0.01) matched closest to γ-Proteobacteria. Although this sequence grouped to the well-studied Enterobacteriaceae family, it did not match any sequence in Genbank by more than 97%. Our observations suggest that abnormal patterns of microbiota and potentially a novel pathogen contribute to the etiology of NEC.     

Probiotics and neonatal necrotizing enterocolitis

Neonatal necrotizing enterocolitis (NEC) is one of few diseases for which probiotics have appeared to have clear benefit in clinical trials, however safety concerns persist. Clinical trials of probiotics have preceded our understanding of the effect of probiotics on the developing gut and microbial colonization patterns of the preterm infant. Colonization of the preterm intestine begins with the birthing process and is then influenced by the neonatal intensive care unit and iatrogenic manipulations. Resulting altered microbiota may have significant implications for the immature preterm gut and susceptibility to NEC.     

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.     

Reduction of experimental necrotizing enterocolitis with anti-TNF-alpha

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. However, despite significant morbidity and mortality, the etiology and pathogenesis of NEC are poorly understood. Evidence suggests that ileal proinflammatory mediators such as IL-18 contribute to the pathology associated with this disease. In addition, we have previously shown that upregulation of TNF-alpha in the liver is correlated with ileal disease severity in a neonatal rat model of NEC. With the use of a neonatal rat model of NEC, we evaluated the incidence and severity of ileal damage along with the production of both hepatic and ileal proinflammatory cytokines in animals injected with (anti-TNF-alpha; n = 23) or without (NEC; n = 25) a monoclonal anti-TNF-alpha antibody. In addition, we assessed changes in apoptosis and ileal permeability in the NEC and anti-TNF-alpha groups. Ileal damage was significantly decreased, and the incidence of NEC was reduced from 80% to 17% in animals receiving anti-TNF-alpha. Hepatic TNF-alpha and hepatic and ileal IL-18 were significantly decreased in pups given anti-TNF-alpha compared with those sham injected. In addition, ileal luminal levels of both TNF-alpha and IL-18 were significantly decreased in the anti-TNF-alpha-injected group. Ileal paracellular permeability and the proapoptotic markers Bax and cleaved caspase-3 were significantly decreased in the anti-TNF-alpha group. These data show that hepatic TNF-alpha is an important component for the development of NEC in the neonatal rat model and suggest that anti-TNF-alpha could be used as a potential therapy for human NEC.     

Interferon-alpha reduces the development of experimental necrotizing enterocolitis

Cytokines had important role in the pathogenesis of necrotizing enterocolitis (NEC). The aim of this study is to investigate if IFN-alpha has a prophylaxic effect on experimental NEC development in newborn rat pups. Twenty eight Wistar Albino newborn rat pups were divided into three groups. Control group rats were breast-fed, NEC group and interferon (IFN) group rat pups were hand-fed with premature newborn formula. IFN-alpha was administered subcutaneously at a dose of 50,000 IU/rat/day in IFN group. NEC was induced experimentally by cold stress twice a day in IFN and NEC groups. On the fourth day, the rats were killed, and all the intestine were removed to determine the tissue level of malonaldehyde (MDA) and histologic changes. The microscopic lesions in the NEC group rats were virtually the same as those seen in neonatal NEC, with severe separation of submucosa and/or lamina propria, loss of villi and in some cases necrosis to extention the muscularis. In contrast, in the rats treated with IFN, lesions were moderate separation of submucosa and/or lamina propria, edema in submucosal and muscular layers. Intestinal injury score and MDA levels in NEC group were significantly higher than in the IFN group (P<0.05). In conclusion it was suggested that IFN-alpha was effective in reducing the severity of NEC in rat pups.     

Nitrosative stress in an animal model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a disease of newborns characterized by gut barrier failure. We reasoned that upregulation of inducible nitric oxide synthase (iNOS) may result in nitrosative stress and accumulation of nitroso species in the intestine. Newborn rats were either breast-fed (BF), or formula-fed and additionally subjected to hypoxia (FFH). At Day 4 after birth, the distal ilea were harvested and processed for Western blot analysis and measurement of NO-related metabolites. While BF neonates showed normal morphology, FFH neonates developed signs of NEC by Day 4. These pathological changes correlated with upregulation of iNOS and increases in tissue nitrite, nitrosothiol, and nitrosamine concentrations. Enhanced nitroso levels were most prominent in the mucosal layers of the ileum and iNOS inhibition resulted in a significant decrease in both nitroso species and incidence of NEC. In contrast, increased nitrite levels were distributed evenly throughout the ileum and remained unchanged following iNOS inhibition. Similarly, specimens from NEC patients had higher intestinal levels of NO-related metabolites compared to non-NEC controls. This is the first report of tissue levels of nitroso species in the gut of an animal model of NEC and of human specimens. The results suggest that local nitrosative stress contributes to the pathology associated with NEC. Unexpectedly, the NO breakdown product nitrite, previously considered biologically inert, was found to be present throughout the ileal wall, suggesting that cellular NO metabolism is altered significantly in NEC. Whether nitrite plays a protective or deleterious role remains to be investigated.     

益生菌预防早产儿坏死性小肠结肠炎的研究进展

新生儿坏死性小肠结肠炎(Neonatal necrotizing enterocolitis,NEC)是早产儿中发病率高、预后差、死亡率高的严重肠道疾病;目前,本病的发病机制尚未明确,缺乏有效的治疗方法。近十几年,大量试验显示,口服益生菌能有效预防早产儿NEC,但其作用机制尚未完全明确,且在益生菌种类选择、给药方式(单药、联合给药)、疗效、安全性等方面,仍存在一些问题。因此,本文就上述问题进行综述。     

金双歧预防新生儿坏死性小肠结肠炎的效果探讨

目的比较金双歧、妈咪爱预防新生儿坏死性小肠结肠炎(NEC)的效果。方法将我院新生儿科2012年7月至2014年6月两年间收治的新生儿随机分为金双歧组、妈咪爱组、对照组。将颅内出血Ⅲ-Ⅳ级、严重先天性疾病的患儿排除。金双歧组给予金双歧口服或鼻饲0.5 g tid,妈咪爱组给予妈咪爱口服或鼻饲1.0 g bid,对照组给予生理盐水口服或鼻饲1.0 mL bid,观察各组NEC发生率。结果金双歧组170例,发生NEC 4例,发生率为2.35%;妈咪爱组160例,发生NEC 10例,发生率为6.25%;对照组150例,发生NEC 20例,发生率为13.33%。结论口服益生菌对预防新生儿NEC有显著作用,可以降低其发病率。金双歧、妈咪爱对预防新生儿NEC差异无统计学意义,但金双歧组的发病率更低,秩和值更低。推荐临床上尽早使用金双歧预防NEC的发生。     

Hepatic inflammatory mediators contribute to intestinal damage in necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Along with pathological effects in the ileum, severe NEC is often accompanied by multisystem organ failure, including liver failure. The aim of this study was to determine the changes in hepatic cytokines and inflammatory mediators in experimental NEC. The well-established neonatal rat model of NEC was used in this study, and changes in liver morphology, numbers of Kupffer cells (KC), gene expression, and histological localization of IL-18, TNF-alpha, and inducible nitric oxide synthase were evaluated. Intestinal luminal TNF-alpha levels were also measured. Production of hepatic IL-18 and TNF-alpha and numbers of KC were increased in rats with NEC and correlated with the progression of intestinal damage during NEC development. Furthermore, increased levels of TNF-alpha in the intestinal lumen of rats with NEC was significantly decreased when KC were inhibited with gadolinium chloride. These results suggest an important role of the liver and the gut-liver axis in NEC pathogenesis.     

Decreased development of necrotizing enterocolitis in IL-18-deficient mice

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease predominantly of prematurely born infants, characterized in its severest from by extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. Proinflammatory cytokines have been implicated in the development of NEC, and we have previously shown that IL-18 is significantly elevated in the well-established neonatal rat model of NEC. To determine whether IL-18 contributes to intestinal pathology in NEC, we subjected IL-18 knockout mice to the protocol used to develop experimental NEC in newborn rats. Newborn B6.129P2-Il18(tm1Aki)/J (NEC IL-18(-/-)) and wild-type (NEC WT) mice were hand fed every 3 h with cow's milk-based formula and exposed to asphyxia and cold stress twice daily. After 72 h, animals were killed and distal ileum and liver were removed. Disease development was determined via histological changes in the ileum as scored by a blinded evaluator. The number of TNF-alpha-, IL-12-, and IL-1beta-positive cells and macrophages were determined in both ileum and liver via immunohistology. IkappaB-alpha and IkappaB-beta were determined from protein extracts from both ileum and liver using Western blot analysis. The incidence and severity of NEC was significantly reduced in NEC IL-18(-/-) mice compared with NEC WT. Furthermore, mean ileal macrophages and hepatic IL-1beta were significantly reduced in IL-18(-/-) mice subjected to the NEC protocol. There were no statistically significant changes in Kupffer cells, hepatic TNF-alpha, ileal IL-1beta, or IL-12. IkappaB-alpha and IkappaB-beta were significantly increased in NEC IL-18(-/-) mice ileum and liver, respectively. These results confirm that IL-18 plays a crucial role in experimental NEC pathogenesis.     

Intestinal post-ischemic reperfusion injury: studies with neonatal necrotizing enterocolitis

In the feline intestine studies have implicated superoxide (O.-) and other oxygen derived free radicals as initiators of injury as measured by increased capillary permeability during the reperfusion period. Biochemical mechanisms of this free radical generation include: xanthine oxidase dependent O.- production, hydrogen peroxide (H2O2) formation by superoxide dismutase (SOD), hydroxyl radical (OH-) production via the Haber-Weiss reaction, and lipid radical formation from membrane peroxidation. Pathological consequences of these events include inflammatory neutrophil infiltration, damage to the collagen and mucosal basement membrane, increased capillary permeability, edema, cell degeneration and necrosis. Animal models of neonatal necrotizing enterocolitis (NNEC) indicate that intestinal injury occurs after the etiologic factors (hypothermia, hypoxia) are removed. In order to determine the role of active oxygen species in the pathogenesis of NNEC, weanling hamsters and neonatal piglets were cold stressed and activities of pro/antioxidant enzymes were determined, and histopathologic and ultrastructural studies were performed. Cold stressed weanling hamsters showed a 55.7% (P less than 0.05) decrease in xanthine dehydrogenase/xanthine oxidase activity ratio. Light microscopy revealed scattered colonic mucosal erosions and submucosal edema in 50% of cold stressed animals. Transmission electron microscopy demonstrated degeneration of colonic mucosal epithelial cells, enlarged intracellular spaces, cytoplasmic vacuolization, and nuclear membrane swelling. The colonic serosa was also edematous and infiltrated with bacteria. Large intestinal tissue from cold stressed neonatal piglets showed a significant increase (P less than 0.05) in Mn and Cu, Zn, SOD, CAT, GSH-Red, total GSH, and Glc6-PD at 0 and 12 hrs. post stress.(ABSTRACT TRUNCATED AT 250 WORDS)     

新生鼠坏死性小肠结肠炎肠道菌群变化及意义

目的探讨新生鼠坏死性小肠结肠炎(NEC)时肠道菌群变化,旨在阐明肠道菌群在NEC发病中的作用,为探讨新生儿NEC的发病机制及寻求有效的防治措施提供理论依据。方法SD新生大鼠出生48 h开始给予鼠乳代用品人工喂养,100%氮气缺氧90 s,4℃冷刺激10 min,每天2次,连续3 d,建立新生SD大鼠NEC模型。40只新生SD大鼠随机分成NEC模型组(A组)和正常对照组(B组)。每组动物各20只。在最后一次缺氧、冷刺激后24 h空腹断头处死大鼠,留取回盲部近端肠管组织进行肠组织损伤评分,组织学评分≥2确定为NEC;实验前后留取两组新生鼠粪便,按照张秀荣方法进行肠道菌群检测。实验前后比较采用配对t检验,组间比较采用方差分析,α=0.05为显著性检验标准。结果模型组新生鼠相继出现腹泻、腹胀、萎靡、活动减少,生长减慢,对照组新生大鼠进食及排便均正常,无腹胀及胃潴留,活动度良好,皮下脂肪丰满。模型组新生大鼠NEC的发生率为100%(20/20),对照组无1例发生NEC。实验组和对照组肠损伤病理评分(x±s)分别为:3.25±0.85、0.45±0.51,t=12.622,P<0.01。模型组和对照组新生鼠实验前肠道细菌总数,杆菌、球菌总数,G 杆菌、G 球菌,G-杆菌、G-球菌数差异均无显著性,肠道菌群中杆菌和球菌的比例都在正常范围中。实验结束时,正常对照组新生大鼠肠道菌群总数明显增多,其中以G 杆菌增加为主;G-杆菌属及G 球菌属菌数占肠道菌群的比率在实验前后差异无显著性;模型组新生大鼠肠道群总数亦明显增多,而且明显高于正常对照组。实验结束时肠道菌群数量,其中主要是G 球菌显著增加,而G 杆菌却较对照组明显减少,与实验前相比,明显下降,差异有显著性;模型组实验结束时肠道菌群中杆菌和球菌比值减少、倒置。结论NEC发病前正常肠道菌群已发生质和量的变化,肠道菌群紊乱在NEC发病机制中起关键作用;及时纠正肠道菌群紊乱可能会降低新生大鼠发生NEC危险性。     

Community analysis of bacteria colonizing intestinal tissue of neonates with necrotizing enterocolitis

Background  

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in newborn neonates. Bacteria are believed to be important in the pathogenesis of NEC but bacterial characterization has only been done on human faecal samples and experimental animal studies. The aim of this study was to investigate the microbial composition and the relative number of bacteria in inflamed intestinal tissue surgically removed from neonates diagnosed with NEC (n = 24). The bacterial populations in the specimens were characterized by laser capture microdissection and subsequent sequencing combined with fluorescent in situ hybridization (FISH), using bacterial rRNA-targeting oligonucleotide probes.     

Role of lactadherin in intestinal barrier integrity in experimental neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is one of the most widespread and devastating gastrointestinal diseases in neonates. Destruction of the intestinal barrier is the main underlying cause of NEC. The aim of this study was to determine the role of lactadherin in preventing NEC in a neonatal rat model and investigate the molecular mechanism of lactadherin-mediated protection of the intestinal barrier. Neonatal rats were divided into three groups: dam feeding (DF), NEC (NEC), and NEC supplemented with 10 μg/(g·day) recombinant human lactadherin (NEC+L). Intestinal permeability, tissue damage, and cell junction protein expression and localization were evaluated. We found that lactadherin reduced weight loss caused by NEC, reduced the incidence of NEC from 100% to 46.7%, and reduced the mean histological score for tissue damage to 1.40 compared with 2.53 in the NEC group. Intestinal permeability of lactadherin-treated rats was significantly reduced when compared with that of the NEC group. In addition, the expression levels of JAM-A, claudin 3, and E-calcium in the ileum of NEC group animals increased compared with those in the ileum of DF group animals, and these levels decreased in the NEC+L group. Lactadherin changed the localization of claudin 3, occludin, and E-cadherin in epithelial cells. The mechanism underlying lactadherin-mediated protection of the intestinal barrier might be restoring the correct expression levels and localization of tight junction and adherent junction proteins. These findings suggest a new candidate agent for the prevention of NEC in newborns.     

酪酸梭菌预防早产儿坏死性小肠结肠炎的疗效观察

目的探讨宝乐安（酪酸梭菌CGMCC0313．1散剂）预防早产儿坏死性小肠结肠炎（NEC）的疗效。方法将胎龄在28—35周的早产几90例,随机分为预防组和对照组,预防组在原发病治疗的基础上出生后即喂服宝乐安,500mg／次,2次／d;对照组仅予原发病治疗。全部早产儿观察NEC的发病率及轻重程度、病程时间、NEC并发肺部感染及早产儿体重增长情况,予大便培养进行病原菌的检测。结果预防组的早产儿在NEC的发病率及轻重程度、病程时间、NEC并发肺部感染、体重增长、病原菌克雷伯菌及大肠埃希菌检出情况等方面与对照组的早产儿比较,差异均具有显著性（P〈0．05）。结论早期服用宝乐安可以降低早产儿坏死性小肠结肠炎的发病率,并能缩短NEC的病程,减轻NEC病情,降低NEC并发肺部感染的发病率,有助于早产儿体重的恢复增长。     

微生态制预防治早产儿坏死性小肠结肠炎的作用研究

目的 观察应用微生态制剂(金双歧)预防早产儿坏死性小肠结肠炎(NEC)的有效性.方法 2011年1月至2012年6月在成都市妇女儿童中心医院新生儿科住院治疗的279例生后存活7d并且开始胃肠道喂养的早产儿,预防组142例,对照组137例.预防组在对照组常规治疗原发病基础上每日给予双歧三联活菌片,每次0.5片,每日3次,用药至出院.观察治疗期间两组发生NEC的情况,对两组治疗前及治疗后7d分别留取新鲜粪便标本进行细菌学检查.结果 预防组NEC发病率为1.41％,对照组NEC发生率为5.84％,两组比较差异有统计学意义(P＜0.05).治疗后7d两组患儿肠道细菌总数、杆菌、球菌总数及杆球菌比值差异均有统计学意义(P＜0.05).预防组治疗前、治疗后7d杆球菌比值差异无统计学意义(P＞0.05).结论 给予早产儿预防性口服微生态制剂能够降低NEC发病率.其机制可能与口服微生态制剂后促进新生儿肠道正常菌群的定植和优势化、维持肠道正常菌群比率有关.