Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Although end-stage NEC is characterized histopathologically as extensive necrosis, apoptosis may account for the initial loss of epithelium before full development of disease. We have previously shown that epidermal growth factor (EGF) reduces the incidence of NEC in a rat model. Although EGF has been shown to protect intestinal enterocytes from apoptosis, the mechanism of EGF-mediated protection against NEC is not known. The aim of this study was to investigate if EGF treatment elicits changes in expression of apoptotic markers in the ileum during the development of NEC. With the use of a well-established neonatal rat model of NEC, rats were divided into the following three experimental groups: dam fed (DF), milk formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC+EGF). Changes in ileal morphology, gene and protein expression, and histological localization of apoptotic regulators were evaluated. Anti-apoptotic Bcl-2 mRNA levels were markedly reduced and pro-apoptotic Bax mRNA levels were markedly elevated in the NEC group compared with DF controls. Supplementation of EGF into formula significantly increased anti-apoptotic Bcl-2 mRNA, whereas pro-apoptotic Bax was significantly decreased. The Bax-to-Bcl-2 ratio for mRNA and protein was markedly decreased in NEC+EGF animals compared with the NEC group. The presence of caspase-3-positive epithelial cells was markedly reduced in EGF-treated rats. These data suggest that alteration of the balance between pro-and anti-apoptotic proteins in the site of injury is a possible mechanism by which EGF maintains intestinal integrity and protects intestinal epithelium against NEC injury.     

Nitrosative stress in plants

Nitrosative stress has become a usual term in the physiology of nitric oxide in mammalian systems. However, in plants there is much less information on this type of stress. Using olive leaves as experimental model, the effect of salinity on the potential induction of nitrosative stress was studied. The enzymatic l-arginine-dependent production of nitric oxide (NOS activity) was measured by ozone chemiluminiscence. The specific activity of NOS in olive leaves was 0.280nmol NOmg(-1) proteinmin(-1), and was dependent on l-arginine, NADPH and calcium. Salt stress (200mM NaCl) caused an increase of the l-arginine-dependent production of nitric oxide (NO), total S-nitrosothiols (RSNO) and number of proteins that underwent tyrosine nitration. Confocal laser scanning microscopy analysis using either specific fluorescent probes for NO and RSNO or antibodies to S-nitrosoglutathione and 3-nitrotyrosine, showed also a general increase of these reactive nitrogen species (RNS) mainly in the vascular tissue. Taken together, these findings show that in olive leaves salinity induces nitrosative stress, and vascular tissues could play an important role in the redistribution of NO-derived molecules during nitrosative stress.     

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。     

The protective and anti-inflammatory effects of glucagon-like peptide-2 in an experimental rat model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease, that affects premature infants. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic hormone and reduces the inflammation. We suspected that GLP-2 would have protective and anti-inflammatory effects in an experimental rat model of NEC. NEC was induced in newborn rats by enteral feeding with hyperosmolar formula, asphyxial stress and enteral administration of lipopolysaccharide (LPS). Rats were randomly divided into the following four groups: dam-fed, NEC, NEC + GLP-2(L) given 80 μg/kg/day of GLP-2, and NEC + GLP-2(H) given 800 μg/kg/day of GLP-2. GLP-2 was administered subcutaneously every 6 h before stress. All animals surviving beyond 96 h or any that developed signs of distress were euthanized. The clinical sickness score in the NEC + GLP-2(H) group was significantly lower than that in the NEC group. The NEC score and the survival rate in the NEC + GLP-2(H) group was significantly improved compared with those in the NEC and the NEC + GLP-2(L) groups. Villous height and crypt depth in both the GLP-2 treatment groups were significantly increased compared with those in the NEC group. There were no significant differences in the crypt cell proliferation indices among the groups. Ileal interstitial TNF-α and IL-6 level in the NEC + GLP-2(H) group was decreased to the same levels in the dam-fed group. High dose GLP-2 administration improved the incidence and survival rate for NEC. It also decreased mucosal inflammatory cytokine production. These results support a potential therapeutic role for GLP-2 in the treatment 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.     

Pomegranate seed oil reduces intestinal damage in a rat model of necrotizing enterocolitis

Pomegranate seed oil (PSO), which is the major source of conjugated linolenic acids such as punicic acid (PuA), exhibits strong anti-inflammatory properties. Necrotizing enterocolitis (NEC) is a devastating disease associated with severe and excessive intestinal inflammation. The aim of this study was to evaluate the effects of orally administered PSO on the development of NEC, intestinal epithelial proliferation, and cytokine regulation in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), formula-fed rats (FF), or rats fed with formula supplemented with 1.5% of PSO (FF + PSO). All groups were exposed to asphyxia/cold stress to induce NEC. Intestinal injury, epithelial cell proliferation, cytokine production, and trefoil factor 3 (Tff3) production were evaluated in the terminal ileum. Oral administration of PSO (FF+PSO) decreased the incidence of NEC from 61 to 26%. Feeding formula with PSO improved enterocyte proliferation in the site of injury. Increased levels of proinflammatory IL-6, IL-8, IL-12, IL-23, and TNF-α in the ileum of FF rats were normalized in PSO-treated animals. Tff3 production in the FF rats was reduced compared with DF but not further affected by the PSO. In conclusion, administration of PSO protects against NEC in the neonatal rat model. This protective effect is associated with an improvement of intestinal epithelial homeostasis and a strong anti-inflammatory effect of PSO on the developing intestinal mucosa.     

The calm mouse: an animal model of stress reduction

Chronic stress is associated with negative health outcomes and is linked with neuroendocrine changes, deleterious effects on innate and adaptive immunity, and central nervous system neuropathology. Although stress management is commonly advocated clinically, there is insufficient mechanistic understanding of how decreasing stress affects disease pathogenesis. Therefore, we have developed a "calm mouse model" with caging enhancements designed to reduce murine stress. Male BALB/c mice were divided into four groups: control (Cntl), standard caging; calm (Calm), large caging to reduce animal density, a cardboard nest box for shelter, paper nesting material to promote innate nesting behavior, and a polycarbonate tube to mimic tunneling; control exercise (Cntl Ex), standard caging with a running wheel, known to reduce stress; and calm exercise (Calm Ex), calm caging with a running wheel. Calm, Cntl Ex and Calm Ex animals exhibited significantly less corticosterone production than Cntl animals. We also observed changes in spleen mass, and in vitro splenocyte studies demonstrated that Calm Ex animals had innate and adaptive immune responses that were more sensitive to acute handling stress than those in Cntl. Calm animals gained greater body mass than Cntl, although they had similar food intake, and we also observed changes in body composition, using magnetic resonance imaging. Together, our results suggest that the Calm mouse model represents a promising approach to studying the biological effects of stress reduction in the context of health and in conjunction with existing disease models.     

Nitrosative stress induced cytotoxicity in Giardia intestinalis

AIMS: To investigate the antigiardial properties of the nitrosating agents: sodium nitrite, sodium nitroprusside and Roussin's black salt. METHODS AND RESULTS: Use of confocal laser scanning microscopy and flow cytometry indicated permeabilization of the plasma membrane to the anionic fluorophore, DiBAC4(3) [bis(1,3-dibutylbarbituric acid) trimethine oxonol]. Loss of plasma membrane electrochemical potential was accompanied by loss of regulated cellular volume control. Changes in ultrastructure revealed by electron microscopy and capacity for oxygen consumption, were also consequences of nitrosative stress. Roussin's black salt (RBS), active at micromolar concentrations was the most potent of the three agents tested. CONCLUSIONS: These multitargeted cytotoxic agents affected plasma membrane functions, inhibited cellular functions in Giardia intestinalis and led to loss of viability. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrosative damage, as an antigiardial strategy, may have implications for development of chemotherapy along with suggesting natural host defence mechanisms.     

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.     

Fecal filtrate transplantation protects against necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a life-threatening gastrointestinal disorder afflicting preterm infants, which is currently unpreventable. Fecal microbiota transplantation (FMT) is a promising preventive therapy, but the transfer of pathogenic microbes or toxic compounds raise concern. Removal of bacteria from donor feces by micropore filtering may reduce this risk of bacterial infection, while residual bacteriophages could maintain the NEC-preventive effects. We aimed to assess preclinical efficacy and safety of fecal filtrate transplantation (FFT). Using fecal material from healthy suckling piglets, we compared rectal FMT administration (FMT, n = 16) with cognate FFT by either rectal (FFTr, n = 14) or oro-gastric administration (FFTo, n = 13) and saline (CON, n = 16) in preterm, cesarean-delivered piglets as models for preterm infants. We assessed gut pathology and analyzed mucosal and luminal bacterial and viral composition using 16S rRNA gene amplicon and meta-virome sequencing. Finally, we used isolated ileal mucosa, coupled with RNA-Seq, to gauge the host response to the different treatments. Oro-gastric FFT completely prevented NEC, which was confirmed by microscopy, whereas FMT did not perform better than control. Oro-gastric FFT increased viral diversity and reduced Proteobacteria relative abundance in the ileal mucosa relative to control. An induction of mucosal immunity was observed in response to FMT but not FFT. As preterm infants are extremely vulnerable to infections, rational NEC-preventive strategies need incontestable safety profiles. We show in a clinically relevant animal model that FFT, as opposed to FMT, efficiently prevents NEC without any recognizable side effects.Subject terms: Bacteriophages, Microbial ecology, Inflammatory bowel disease     

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.     

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.     

Oxidative stress in a phenylketonuria animal model

Oxidative stress is seen in various metabolic disorders for unknown reasons. Oxidative stress is defined as an imbalance between pro-oxidant and antioxidant status in favor of the former. This study investigated whether oxidative stress exists in phenylketonuria (PKU) using the BTBR-Pah(enu2) animal model for PKU. Animals (14-24 weeks old) were sacrificed and brain and red blood cells (RBCs) were obtained aseptically. The lipid peroxidation by-product, evaluated as malondialdehyde (MDA), was significantly higher in the brains and RBCs of PKU animals (n = 6) than in controls (n = 6). Glutathione/glutathione disulfide, a good indicator for tissue thiol status, was significantly decreased both in the brains and RBCs. Some antioxidant enzymes were also analyzed in RBCs, including glucose-6-phosphate dehydrogenase (G6PD), which provides the RBC's main reducing power, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and catalase detoxifies H2O2 by catalyzing its reduction to O2 and H2O. Both catalase and G6PD were significantly increased in the RBCs of PKU animals.     

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.     

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。     

双歧杆菌对新生大鼠坏死性小肠结肠炎肠损伤及肠细胞凋亡影响

目的探讨添加双歧杆菌对新生鼠坏死性小肠结肠炎(necrotizing enterocolitis,NEC)模型肠损伤的保护作用。方法 32只新生SD大鼠按析因设计随机分成4组,每组动物8只。A1B1组为NEC模型组并添加双歧杆菌(109CFU/d),A1B2组为NEC模型组,但未添加双歧杆菌;A2B1组为对照组并添加双歧杆菌(109CFU/d),A2B2组为对照组,且未添加双歧杆菌。在出生48 h开始给予鼠配方奶人工喂养,100%氮气缺氧90 s,4℃冷刺激10 min,每天2次,连续3 d,建立新生大鼠NEC模型;在最后1次缺氧、冷刺激后24 h空腹断头处死小鼠,解剖留取十二指肠下端至直肠上端肠道组织,其中,回盲部近端肠管进行病理学检查及肠损伤评分,组织学评分≥2为NEC,其余肠管进行肠细胞凋亡率检测及电镜观察。采用流式细胞仪检测肠细胞凋亡率。SPSS 11.0统计学软件进行统计分析,α=0.05为显著性检验标准。结果造模后,A1B1组、A1B2组相继出现腹泻、腹胀、生长发育减慢和活动度减少,显微镜下可见肠黏膜坏死、黏膜下层出血以及肌肉层坏死等肠损伤表现,透射电镜显示肠黏膜出现大量凋亡细胞,形成凋亡小体,但A1B1组程度较轻。A1B1、A1B2、A2B1和A2B24组肠损伤组织病理评分(x±s)分别为2.04±0.52、3.38±0.55、0.33±0.36和0.38±0.33,肠细胞凋亡率分别为(23.97±10.48)%、(47.28±21.98)%、(11.42±4.75)%和(12.16±4.95)%;各组间肠损伤组织评分、肠细胞凋亡率差异有显著统计学意义(H分别为26.657、20.916,P均0.01);与A1B2组相比,A1B1组新生鼠肠损伤组织评分、肠细胞凋亡率均明显降低,但仍高于A2B1、A2B22个对照组(P均0.01);肠组织损伤评分值、肠细胞凋亡率均受到NEC造模和添加双歧杆菌两个因素的影响,NEC造模与补充双歧杆菌之间均存在交互作用。结论添加双歧杆菌可以降低新生鼠NEC肠损伤程度,可能通过抑制肠上皮细胞凋亡,从而降低新生大鼠发生NEC危险性。