Rat Model Of Necrotizing Enterocolitis Research Articles

Identification and evaluation of probiotic potential of Bifidobacterium breve AHC3 isolated from chicken intestines and its effect on necrotizing enterocolitis (NEC) in newborn SD rats.

Necrotizing enterocolitis (NEC) is a severe intestinal disease of the newborn infants, associated with high morbidity and mortality. It has been reported that Bifidobacterium could protect the intestinal barrier function and reduce the risk of NEC. This study aimed to evaluate the probiotic potential of Bifidobacterium strains isolated from the chicken intestines and its effect on necrotizing enterocolitis in newborn SD rats. Out of 32 isolates, B. breve AHC3 not only exhibited excellent probiotic potential, including tolerance to artificial simulated gastric conditions, adhesion to HT-29 cells, antioxidant capacity and antibacterial activity, but also possessed reliable safety. Additionally, NEC model was established to further investigate the effect of B. breve AHC3 on necrotizing enterocolitis in newborn SD rats. It was illustrated that administration of B. breve AHC3 significantly not only reduced the incidence of NEC (from 81.25% to 34.38%) (P< 0.05), but also alleviated the severity of ileal injury (P< 0.05). Compared with NEC model, B. breve AHC3 could significantly decrease the level of proinflammatory factor TNF-α (P< 0.05) and increase the level of antiinflammatory factor IL-10 (P< 0.05) in the ileum of NEC rats. Through the intervention of B. breve AHC3, the gray value of inducible nitric oxide synthase (iNOS) in intestinal tissue of NEC rats was significantly reduced (P< 0.05). It was indicated that B. breve AHC3 exhibited prominent probiotic potential and reliable safety. In the neonatal SD rat model of NEC, B. breve AHC3 had an available protective effect on the intestinal injury of NEC, which might be related to reducing the inflammatory reaction in the ileum and inhibiting the expression of iNOS in intestinal tissue cells. B. breve AHC3 could be used as a potential treatment for human NEC.

Effect of gut microbiota homeostasis on hematopoiesis in a neonatal rat model of necrotizing enterocolitis

To study the effect of gut microbiota on hematopoiesis in a neonatal rat model of necrotizing enterocolitis (NEC). Neonatal Sprague-Dawley rats were randomly divided into a control group and a model group (NEC group), with 6 rats in each group. Formula milk combined with hypoxia and cold stimulation was used to establish a neonatal rat model of NEC. Hematoxylin and eosin staining was used to observe the pathological changes of intestinal tissue and hematopoiesis-related organs. Routine blood tests were conducted for each group. Immunohistochemistry was used to observe the changes in specific cells in hematopoiesis-related organs. Flow cytometry was used to measure the changes in specific cells in bone marrow. 16S rDNA sequencing was used to observe the composition and abundance of gut microbiota. Compared with the control group, the NEC group had intestinal congestion and necrosis, damage, atrophy, and shedding of intestinal villi, and a significant increase in NEC histological score. Compared with the control group, the NEC group had significantly lower numbers of peripheral blood leukocytes and lymphocytes (P<0.05), nucleated cells in the spleen, thymus, and bone marrow, and small cell aggregates with basophilic nuclei in the liver (P<0.05). The NEC group had significant reductions in CD71+ erythroid progenitor cells in the liver, CD45+ lymphocytes in the spleen and bone marrow, CD3+ T lymphocytes in thymus, and the proportion of CD45+CD3-CD43+SSChi neutrophils in bone marrow (P<0.05). There was a significant difference in the composition of gut microbiota between the NEC and control groups, and the NEC group had a significant reduction in the abundance of Ligilactobacillus and a significant increase in the abundance of Escherichia-Shigella (P<0.05), which replaced Ligilactobacillus and became the dominant flora. Multi-lineage hematopoietic disorder may be observed in a neonatal rat model of NEC, which may be associated with gut microbiota dysbiosis and abnormal multiplication of the pathogenic bacteria Escherichia-Shigella.

Deciphering the role of TNF‑α‑induced protein8‑like2 in the pathogenesis of necrotizing enterocolitis in neonatal rats.

Neonatal necrotizing enterocolitis (NNEC) is a disease characterized by intestinal inflammation and ischemic necrosis. Despite progress having been made during decades of research, details regarding its pathophysiology remain to be elucidated. It is known that abnormal expressions of TNF-α-induced protein 8-like 2 (TIPE2) can be observed in several diseases. However, the expression of TIPE2 in necrotizing enterocolitis (NEC) rats has not been examined before. The present study aimed to describe the expression pattern of TIPE2 and its role in NNEC pathogenesis. An NEC rat model was generated and used in the present study. All rats were sacrificed when the phenotype developed and the intestine between the lower end of the duodenum and the ileocecal were collected for further study. Hematoxylin and eosin, and immunohistochemical staining, reverse transcription-quantitative PCR and western blotting analysis were used to examine the expression of TIPE2. The results showed that the average body weight was significantly decreased in the NEC group compared with the control group along with a significant decrease of TIPE2 expression. However, the expressions of phosphatidylinositol-3-kinase (PI3K) and serine/threonine kinase AKT were significantly increased in NEC rats. The correlation analysis showed that the expressions of TIPE2 and PI3K were negatively correlated with a correlation coefficient of -0.797. To further determine the association between TIPE2 and PI3K/AKT pathway, two groups of wild type Sprague Dawley rats were infected with recombinant adenovirus Ad-V and Ad-TIPE2 respectively. The results showed that the expression of TIPE2 was significantly increased among rats in the Ad-TIPE2-infected group (OE group) compared to the ones from the Ad-V-infected group (NC group). However, the mRNA and protein expressions of PI3K and AKT were significantly decreased in Ad-TIPE2-infected rates. The difference of each index between OE and NC groups was statistically significant. The present study showed that the expression of TIPE2 was downregulated in NEC rats. TIPE2 might be involved in the pathogenesis of NEC by activating the PI3K/Akt signaling pathway.

Intestine and brain TLR-4 modulation following N-acetyl-cysteine treatment in NEC rodent model

Necrotizing enterocolitis (NEC) brain injury is mediated through Toll-like receptor 4 (TLR4) on the intestinal epithelium and brain microglia. Our aim was to determine whether postnatal and/or prenatal NAC can modify NEC associated intestinal and brain TLR4 expression and brain glutathione levels in a rat model of NEC. Newborn Sprague–Dawley rats were randomized into three groups: Control (n = 33); NEC (n = 32)—hypoxia and formula feeding; and NEC-NAC (n = 34)—received NAC (300 mg/kg IP) in addition to NEC conditions. Two additional groups included pups of dams treated once daily with NAC (300 mg/kg IV) for the last 3 days of pregnancy: NAC-NEC (n = 33) or NAC-NEC-NAC (n = 36) with additional postnatal NAC. Pups were sacrificed on the fifth day, and ileum and brains harvested for TLR-4 and glutathione protein levels. Brain and ileum TLR-4 protein levels were significantly increased in NEC offspring as compared to control (brain 2.5 ± 0.6 vs. 0.88 ± 0.12 U and ileum 0.24 ± 0.04 vs. 0.09 ± 0.01, p < 0.05). When NAC was administered only to dams (NAC-NEC) a significant decrease in TLR-4 levels was demonstrated in both offspring brain (1.53 ± 0.41 vs. 2.5 ± 0.6 U, p < 0.05) and ileum (0.12 ± 0.03 vs. 0.24 ± 0.04 U, p < 0.05) as compared to NEC. The same pattern was demonstrated when NAC was administered only or postnatally. The decrease in brain and ileum glutathione levels observed in NEC offspring was reversed with all NAC treatment groups. NAC reverses the increase in ileum and brain TLR-4 levels and the decrease in brain and ileum glutathione levels associated with NEC in a rat model, and thus may protect from NEC associated brain injury.

Prenatal Heparin-Binding Epidermal Growth Factor–Like Growth Factor Administration Decreases Lung Inflammation in the Experimental Model of Necrotizing Enterocolitis

INTRODUCTION: Necrotizing enterocolitis (NEC) is the leading gastrointestinal cause of death in premature infants. Postnatal enteral and prenatal intraperitoneal Heparin-Binding EGF-like Growth Factor (HB-EGF) administration has decreased the incidence and severity of intestinal injury in a neonatal rat model of NEC. The aim was to determine the effect of prenatally administered HB-EGF on lung injury in neonatal rats exposed to the experimental model of NEC. METHODS: Pregnant rats and pups were separated into 3 groups: control (natural birth, no NEC protocol, no HB-EGF), NEC (cesarean section, NEC protocol), and HB-EGF (prenatal HB-EGF administration, cesarean section, NEC protocol). Pregnant rats were given HB-EGF (800 μg/kg/dose) via intraperitoneal injection 2 hours before timed cesarean section. After delivery, the premature rat pups were exposed to the NEC model (hypercaloric feds, hypothermia, and hypoxia). Pups were sacrificed upon clinical signs of NEC; the intestines and lungs were hematoxylin and eosin stained for grading. RESULTS: The median alveolar wall thicknesses were significantly different between control (71,646 μm2) vs NEC (75,398 μm2; p < 0.0001) and HB-EGF (71,646 μm2) vs NEC (p < 0.0001); however, the control and HB-EGF groups were similar (p = 0.307; Fig. 1A). The median alveolar areas were significantly different between control (85.23 μm2) vs NEC (66.40 μm2; p < 0.0001) and HB-EGF (64.14 μm2) vs NEC (p < 0.0001); however, again, the control and HB-EGF were similar (p = 0.215; Fig. 1B).CONCLUSION: Our results support the prenatal HB-EGF role in preventing lung injury by means of preserving alveolar wall thickness and alveolar surface area in this animal model of NEC; therefore, maternal administration of HB-EGF before delivery in prematurity could protect lungs from NEC-related injury.

Peptide Tat(48-60) YVEEL protects against necrotizing enterocolitis through inhibition of toll-like receptor 4-mediated signaling in a phosphatidylinositol 3-kinase/AKT dependent manner.

Necrotizing enterocolitis (NEC) is a catastrophic disease largely occurring in preterm infants, and toll-like receptor 4 (TLR4) has been implicated in its pathogenesis. The current therapeutic strategies for NEC are, however, far from optimal. In the present study, a whey-derived antioxidative peptide conjugated with a cell-penetrating TAT [Tat (48–60) YVEEL] was prepared to endow it with enhanced cell uptake capability and bioavailability. The protective effect of Tat (48–60) YVEEL on experimental NEC was evaluated both in vitro and in vivo. Inhibition of TLR4-mediated signaling by Tat (48–60) YVEEL was assessed in FHC and IEC-6 enterocytes, neonatal rat model of NEC, and the mechanism underlying this effect was determined. Tat (48–60) YVEEL significantly inhibited TLR4-mediated expression of pro-inflammatory cytokines, p65 nuclear translocation and restored the impaired enterocyte migration in cultured enterocytes. In addition, Tat (48–60) YVEEL administration strikingly increased the survival rate, and reduced the severity of NEC in rats through inhibition of TLR4-mediated signaling. These protective effects of Tat (48–60) YVEEL occurred in a PI3K/AKT dependent manner, as administration of PI3K activator Ys49 abrogated its protective effects. Combined with liposomes, Tat (48–60) YVEEL demonstrated longer retention in the intestines that better for potential clinical applications. These data demonstrate that Tat (48–60) YVEEL protects against NEC through inhibition of TLR4-mediated signaling in a PI3K/AKT dependent manner, and offer a potential therapeutic approach to this disease.

Next-Generation Probiotic Therapy to Protect the Intestines From Injury.

Probiotics are live microorganisms that, when administered in adequate amounts, provide health benefits to the host. Some strains of the probiotic Lactobacillus reuteri (L. reuteri) have both antimicrobial and anti-inflammatory properties that may be exploited for the treatment and prevention of different gastrointestinal diseases, including necrotizing enterocolitis (NEC) and Clostridioides difficile (C. difficile) infection. Our laboratory has developed a new delivery system for L. reuteri in which the probiotic is incubated with biocompatible, semipermeable, porous dextranomer microspheres (DM) that can be loaded with beneficial and diffusible cargo. L. reuteri can be induced to form a biofilm by incubating the bacteria on the surface of these microspheres, which enhances the efficacy of the probiotic. Loading the DM with sucrose or maltose induces L. reuteri to produce more biofilm, further increasing the efficacy of the probiotic. Using a rat model of NEC, L. reuteri administered in its biofilm state significantly increases animal survival, reduces the incidence of NEC, preserves gut barrier function, and decreases intestinal inflammation. In a murine model of Clostridiodes difficile infection, L. reuteri administered in its biofilm state decreases colitis when administered either before or after C. difficile induction, demonstrating both prophylactic and therapeutic efficacy. There are currently no FDA-approved probiotic preparations for human use. An FDA-approved phase I clinical trial of L. reuteri in its biofilm state in healthy adults is currently underway. The results of this trial will be used to support a phase 1 clinical trial in neonates, with the goal of utilizing L. reuteri in its biofilm state to prevent NEC in premature neonates in the future.

LncRNA and mRNA profiles of human milk-derived exosomes and their possible roles in protecting against necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is one of the most severe diseases commonly afflicting premature infants. Our previous studies suggests that human milk-derived exosomes (HM-Exos) have a potential therapeutic effect on NEC. In this study, we investigate the potentially therapeutic role of HM-Exos in an NEC animal model via comprehensive lncRNA and mRNA expression profiles. A rat model of NEC was induced through hypoxia, hypothermia and formula feeds. We extracted exosomes from the colostrum of healthy lactating mothers and identified their functions in an NEC animal model. Furthermore, high-throughput lncRNA and mRNA sequencings were explored to find the underlying mechanisms. Although both exosomes from term human breast milk (Term-Exos) and exosomes from preterm human breast milk (Pre-Exos) alleviated the severity of NEC, Pre-Exos seemed to better promote the proliferation of intestinal epithelial cells in vivo. We identified a total of 44 differentially expressed lncRNAs and 88 differentially expressed mRNAs between Term-Exos and Pre-Exos. Further GO and KEGG pathway analysis showed that the lncRNA-mRNA network of HM-Exos was associated with the JAK-STAT signaling pathway, bile secretion and the AMPK signaling pathway, which are predicted to be involved in the proliferation of cells. Therefore, this study reveals for the first time the important roles of human milk derived lncRNAs and mRNAs in protecting against necrotizing enterocolitis. These results provide new insight into the development of NEC.

MiR‑34a increases inflammation and oxidative stress levels in patients with necrotizing enterocolitis by downregulating SIRT1 expression.

The miR‑34a/SIRT1 signaling axis is an important signaling axis in tumors and diseases. Notably, low SIRT1 expression in the intestinal tissues of patients with necrotizing enterocolitis (NEC) has been reported. However, whether miR‑34a/SIRT1 signaling as a target to protect the intestines during the NEC process is unclear and remains to be elucidated. Blood samples were collected from 30patients with NEC, and an NEC rat model was used. The miR‑34a and SIRT1 gene and protein expression levels were assayed by qPCR and Western blotting method. The inflammatory cytokine levels and oxidative stress levels were detected using the ELISA method. The results demonstrated that birth weight, albumin and glucose concentrations were significantly decreased in the NEC patient group compared with the control group, but the C‑reactive protein (CRP) and procalcitonin (PCT) concentrations were significantly increased. The miR‑34a expression level was notably increased in the NEC group, but the SIRT1 expression level was markedly decreased. Notably, the miR‑34a was significantly correlated with NEC severity and the concentrations of CRP, PCT, IL‑6, TNF‑α, IL‑1β, IL‑8, MCP‑1, VCAM1 and malondialdehyde (MDA), but was significantly negatively correlated with SIRT1 gene expression and the concentration of IL‑10. Intestinal villi damage in NEC rats was decreased with miR‑34a inhibition and SIRT1 activation treatment by decreasing the levels of inflammatory cytokines, including IL‑6, TNF‑α, IL‑1β and IL‑8, and oxidative stress proteins, including MCP‑1, VCAM1, and MDA, as well as increasing the level of the anti‑inflammatory cytokine IL‑10. In addition, the results indicated that miR‑34a inhibition and SIRT1 activation strongly protected the intestine and decreased the damage caused by NEC, not only by decreasing the protein levels of SIRT1, TNF‑α, IL‑1β, IL‑6 and IL‑8, but also by increasing the IL‑10 protein levels. The miR‑34a inhibition and SIRT1 activation may decrease the damage caused by NEC by decreasing proinflammatory cytokines and oxidative stress proteins and by increasing the anti‑inflammatory cytokine pathway. Based on the aforementioned analysis, the miR‑34a and SIRT1 proteins may be potential novel therapeutic targets in NEC.

Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model.

The etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NEC's main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels-as seen during stress-promote NEC in formula-fed (FF) newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure-NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p<0.001). Astressin prevented development of NEC in FF-stressed neonates (7.7% vs. 40.0%; p = 0.003). CRF and CRFR immunoreactivity increased in the ileum of neonates with NEC compared to dam-fed controls or FF unstressed pups. Immunoblotting confirmed increased TLR4 protein levels in FF stressed (NEC model) animals vs. controls, and Astressin treatment restored TLR4 to control levels. Peripheral CRF may serve as specific pharmacologic target for the prevention and treatment of NEC.

Astaxanthin Reduces the Severity of Intestinal Damage in a Neonatal Rat Model of Necrotizing Enterocolitis.

This study aimed to ascertain the effects of astaxanthin (ASX) in an experimental necrotizing enterocolitis (NEC) model using rat pups. Forty-two pups born from five Wistar albino rats were randomly divided into three groups as the control group, NEC + placebo (saline), and NEC + ASX. Pups in the NEC + ASX group were given 100 mg/kg/day oral ASX from day 1 to day 4 of the study. Saline of 2 mL/kg was given to the NEC + placebo group. Histopathological, immunohistochemical (caspase-3), and biochemical evaluations including the total antioxidant status (TAS), total oxidant status (TOS), superoxide dismutase (SOD), glutathione (GSH), lipid hydroperoxide (LPO), 8-hydroxydeoxyguanosine (8-OHdG), advanced oxidation protein products (AOPP), myeloperoxidase (MPO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and nuclear factor erythroid 2-related factor 2 (Nfr-2) activities were all performed. A better survival rate and weight gain were demonstrated in the NEC + ASX group (p < 0.05). In the histopathological evaluation, the severity of intestinal damage was significantly reduced in the NEC + ASX group, as well as decreased apoptosis (enzyme-linked immunosorbent assay [ELISA] for caspase-3; p = 0.001). The biochemical analyses of intestinal tissue TOS, oxidative stress index (OSI; TOS/TAS), IL-1β, LPO, 8-OHdG, AOPP, caspase-3 (p < 0.001 for all), and TNF-α and MPO (p = 0.001 for both parameters) levels were lower in the NEC + ASX group than in the NEC + placebo group. Nrf-2, TAS, GSH, and SOD levels were higher in the NEC + ASX group than in the NEC + placebo group (p = 0.001, 0.001, <0.001, and 0.01, respectively). ASX treatment has been shown to effectively reduce the severity of intestinal damage in NEC due to its antioxidant, anti-inflammatory, and antiapoptotic properties. · NEC causes extremely high morbidity and mortality, as well as many complications.. · We investigated the effectiveness of ASX in the experimental NEC model created in rat pups.. · First study examining the effect of ASX on the experimental NEC rat model..

Urine-derived extracellular vesicle miRNAs as possible biomarkers for and mediators of necrotizing enterocolitis: A proof of concept study

BackgroundEarly-stage symptomology of necrotizing enterocolitis (NEC) is similar in presentation to non-NEC sepsis, though the treatment plans differ based on antibiotic administration and withholding of feeds. Improved diagnostics for NEC differentiation would allow clinicians to more rapidly set individual patients on a targeted treatment path. Extracellular vesicle-derived miRNAs, have previously demonstrated efficacy as disease biomarkers. To determine if these miRNAs are differentially-expressed in NEC infants, we performed transcriptomic analysis of urine-derived extracellular vesicle-derived miRNAs. MethodsUrine was non-invasively obtained from infants in one of four groups (n ≥ 8) (Medical NEC, Surgical NEC, non-NEC sepsis, and healthy age-matched controls). EV-derived miRNAs were isolated and transcriptomic analysis was performed. ResultsMultiple miRNAs, including miR-376a, miR-518a-3p and miR-604, were significantly altered when comparing NEC to non-NEC sepsis and healthy controls, and could potentially be used as specific NEC biomarkers. Additionally, Ingenuity Pathway Analysis demonstrated that miRs differentially-expressed in NEC were associated with inflammatory disease and intestinal disease. Signal transduction molecules associated with NEC including TP53 and RPS15, which were also reduced transcriptionally in a rat model of NEC. ConclusionThese data indicate that there is a pool of potential urine EV-derived miRNAs that may be validated as NEC biomarkers in the differentiation of NEC from non-NEC sepsis and from age-matched controls. Additionally, signal transduction molecules associated with miRNAs differentially-expressed in human NEC are altered in a murine model of NEC, suggesting potential crossover between murine models of the disease and actual human presentation. Level of EvidenceLevel III Study of Diagnostic Test.

Maternal N-acetyl-cysteine prevents neonatal brain injury associated with necrotizing enterocolitis in a rat model.

Preterm infants with necrotizing enterocolitis (NEC) are at increased risk of cerebral injury and neurodevelopmental dysfunction. N-acetyl-cysteine (NAC) is a known anti-inflammatory and antioxidant agent. Currently, there is no prophylactic treatment in clinical use to prevent NEC and its neurodevelopmental sequelae. We sought to determine whether brain inflammation/apoptosis accompanies NEC systemic inflammation, and whether it can be attenuated by maternal NAC treatment during pregnancy and/or in the neonatal period in a rat model. An established NEC newborn model (hypoxia 5% O2 for 10min and formula feeding thrice daily, beginning on day 1 for 4days) was used in Sprague-Dawley rat pups (n=32). An additional group of pups (n=33) received NAC (300mg/kg intraperitoneal thrice daily) in addition to NEC conditions (NEC-NAC). Control pups (n=33) were nursed and remained with the dam in room air. Two additional groups included pups of dams treated once daily with NAC (300mg/kg intravenous) in the last 3days of pregnancy. After birth, pups were randomized into NAC-NEC (n=33) with NEC conditions and NAC-NEC-NAC (n=36) with additional postnatal NAC treatment. Pups were sacrificed on the fifth day of life. Pup serum interleukin (IL)-6 protein levels, and brain nuclear factor kappa B (NF-κB) p65, neuronal nitric oxide synthase (nNOS), Caspase 3, tumor necrosis factor alpha (TNF-α), IL-6 and IL-1β protein levels were determined by ELISA, western blot and TUNEL staining, and the groups were compared using analysis of variance (ANOVA). NEC pups had significantly increased serum IL-6 levels compared with the control group as well as increased neuronal apoptosis and brain protein levels of NF-κB, nNOS, Caspase 3, TNF-α, IL-6 and IL-1β compared with control. In all NAC treatment groups, levels of serum IL-6, neuronal apoptosis and brain NF-κB, nNOS, Caspase 3, TNF-α, IL-6 and IL-1β protein levels were significantly reduced compared with the NEC group. The most pronounced decrease was demonstrated within the NAC-NEC-NAC group. NAC treatment can attenuate newborn inflammatory response syndrome and decrease offspring brain neuroapoptosis and inflammation in a rat model of NEC by inhibition of NF-κB, nNOS and Caspase 3 pathways.

Ursodeoxycholic acid protects neonatal rats from necrotizing enterocolitis: a biochemical, histopathological, and immunohistochemical study

Background The pathophysiology of necrotizing enterocolitis (NEC) includes the massive production of endogenous cytokines with exaggerated activation of inflammatory pathways. Ursodeoxycholic acid (UDCA) has been used as an anti-inflammatory, antioxidant, and anti-apoptotic agent. We investigated the possible protective effects of UDCA in a neonatal rat pup model of NEC. Methods We randomly divided rat pups into three groups: a control group, a non-treated NEC group, and a UDCA-treated NEC group. We induced NEC by feeding formula enterally and via hypoxia/reoxygenation. Intestinal samples were collected for histopathological and immunohistochemical evaluation. Blood samples were taken for biochemical analyses. Results UDCA significantly reduced the extents of terminal ileal and jejunal injuries compared to the NEC group (p < .01), reduced Bax and caspase-3 immunoreactivities (both p < .01), and lowered serum levels of platelet-activating factor and intestinal fatty acid-binding protein (p < .01, p = .023, respectively). Conclusions In a rat model of NEC, UDCA protects against adverse intestinal histological, immunohistochemical, and biochemical changes. UDCA significantly reduces the effects of NEC on the rat pup intestine.

Small RNA Sequencing Reveals Differentially Expressed miRNAs in Necrotizing Enterocolitis in Rats.

Necrotizing enterocolitis (NEC) is the leading cause of death due to gastrointestinal disease in preterm infants. The role of miRNAs in NEC is still unknown. The objective of this study was to identify differentially expressed (DE) miRNAs in rats with NEC and analyze their possible roles. In this study, a NEC rat model was established using Sprague-Dawley rat pups. Small RNA sequencing was used to analyze the miRNA expression profiles in the NEC and control rats. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were carried out to identify target mRNAs for the DE miRNAs and to explore their potential roles. The DE miRNAs were verified by real-time quantitative PCR (RT-qPCR). The status of intestinal injury and the elevated levels of inflammatory cytokines in the NEC group confirmed that the NEC model was successfully established. The 16 miRNAs were found to be differentially expressed between the NEC group and the control group of rats. Bioinformatics analysis indicated that the parental genes of the DE miRNAs were predominantly implicated in the phosphorylation, cell migration, and protein phosphorylation processes. Moreover, the DE miRNAs were mainly found to be involved in the pathways of axon guidance, endocytosis, and focal adhesion, as well as in the Wnt signaling pathway, which is related to colitis. The expression patterns of the candidate miRNAs (rno-miR-27a-5p and rno-miR-187-3p), as assessed by RT-qPCR, were in accordance with the expression patterns obtained by miRNA-sequencing. The miRNA/mRNA/pathway network revealed that rno-miR-27a-5p and rno-miR-187-3p might be involved in NEC via the Wnt signaling pathway. We found an altered miRNA expression pattern in rats with NEC. We hypothesize that rno-miR-27a-5p and rno-miR-187-3p might mediate the NEC pathophysiological processes via the Wnt signaling pathway.

Human breast milk oligosaccharides attenuate necrotizing enterocolitis in rats by suppressing mast cell accumulation, DPPI activity and TLR4 expression in ileum tissue, and regulating mitochondrial damage of Caco-2 cells

Necrotizing enterocolitis (NEC), a devastating infant disease characterized by severe intestinal necrosis, its pathogenesis is poorly understood, but appears to be multifactorial and highly associated with immaturity of gastrointestinal tract and immature innate-immune system. Breast-milk is effective strategy to protect infants against NEC. This study is using a NEC rat model to investigate the pathological mechanism of NEC involved intestinal-damages, and the therapeutic mechanism of sialylated human milk oligosaccharides (SHMOs) on NEC rats; also using cell model to investigate the effects of SHMOs on colon-epithelial cells (Caco-2) in-vitro.Extraction and characterization of SHMOs from breast milk, establishment of a NEC rat model, histopathological analysis and mast cell accounting of the terminal ileum were taken; The levels of DPPI, TLR4, IL-6, TNF-α, MMP-2/9 and glutathione were measured using various methods. Caco-2 cells were pre-treated with SHMOs and cultured with LPS, histamine, chymase or DPPI, cell viabilities and mitochondrial membrane potential were examined; flow cytometry was used to detect cell cycle.The accumulation of mast cells was found in the ileum of NEC rats, but prohibited by SHMOs treatment; the increased levels of TLR4, DPPI, IL-6, TNF-α, MMP-2/9 in NEC ileum were suppressed by SHMOs in-vivo. SHMOs prevented Caco-2 cells from LPS, histamine, chymase induced damages by surviving cell viability, regulating G0/G1 and S phase in cell cycles, and increasing mitochondrial membrane potential. These findings provide a new insight into the pharmacological mechanism of SHMOs treatment for NEC and suggest that SHMOs needs well attention for therapeutic aims.

Inhibition of A2B Adenosine Receptor Attenuates Intestinal Injury in a Rat Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a lethal gastrointestinal tract disease that occurs in premature infants. Adenosine receptor A2B (A2BR) regulates the inflammation cytokine secretion and immune cell infiltration in the colonic pathophysiology conditions. In the present study, we aim to determine the roles of A2BR in the development of NEC. A NEC rat model was established and treated with A2BR agonist-BAY60-6583 or A2BR antagonist-PSB1115. Animals in the control group were free from any interventions. Our results showed that the inhibition of A2BR PSB1115 improved intestinal injury and inflammation in newborn NEC rats. The expression levels of caspase-3 and the ratio of apoptotic cells were upregulated in NEC rats, and these indices were downregulated after treating with PSB1115 but further upregulated by BAY60-6583. Meanwhile, a similar trend was also witnessed in the changes of MPO activities and proinflammatory cytokines including IL-6, IFN-γ, and TNF-α. However, the anti-inflammatory cytokine IL-10 in the NECP group was significantly higher than that in the NEC and NECB groups (p < 0.05, respectively). Moreover, the expression of Ki67 was significantly increased in the NECP group as compared with those of the NEC and the NECB groups (p < 0.05, respectively). Collectively, our study suggested that the inhibition of A2BR attenuates NEC in the neonatal rat, at least partially through the modulation of inflammation and the induction of epithelial cell proliferation.

Cronobacter sakazakii induces necrotizing enterocolitis by regulating NLRP3 inflammasome expression via TLR4.

Introduction. Neonatal infection with Cronobacter sakazakii can cause severe intestinal damage and necrotizing enterocolitis (NEC). The inflammasome and Toll-like receptors mediate intestinal damage caused by other intestinal pathogens causing NEC, but the exact mechanism is unclear.Aim. We evaluated the molecular mechanisms underlying C. sakazakii-induced NEC.Methodology. The effects of C. sakazakii treatment on two cell lines and a Sprague-Dawley rat model of NEC were evaluated by a cell death assay, western blot and real-time PCR analyses of the NLRP3 inflammasome and downstream factors, and observation of cell and intestinal damage.Results. C. sakazakii caused cellular damage in vitro, as well as intestinal damage in an animal model. NLRP3, caspase-1, TLR4 and MyD88, as well as the downstream factor IL-1β, were upregulated in C. sakazakii-infected J774A.1 and HT-29 cells. Western blotting showed that C. sakazakii-infected J774A.1 and HT-29 cells and the NEC rat model had higher expression levels of N-terminal gasdermin D (GSDMD) compared with those in the control groups. C. sakazakii and its components promote NF-κB expression via the TLR4/MyD88 signalling pathway, thereby regulating the NLRP3 inflammasome and mediating GSDMD cleavage, resulting in pyroptosis-induced intestinal damage.Conclusion. We found that C. sakazakii upregulates NF-κB via TLR4/MyD88 to promote activation of the NLRP3 inflammasome, leading to the up-regulation of downstream caspase-1, release of IL-1β, GSDMD-mediated pyroptosis and development of NEC. These findings clarify the mechanisms by which C. sakazakii contributes to NEC.

Profile analysis reveals endogenous RNAs regulate necrotizing enterocolitis progression

Necrotizing enterocolitis (NEC) is one of the most common and devastating gastrointestinal diseases in preterm newborns, and its underlying mechanisms remain unclear. Non-coding RNAs (ncRNAs) play critical roles in intestinal diseases; however, little is known about their roles in the development of NEC. To gain a deeper understanding of the pathophysiological mechanism of NEC, long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs were detected in an NEC rat model. In total, 1820 lncRNAs, 118 miRNAs and 929 mRNAs were differentially expressed in NEC group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that these molecules were enriched in apoptosis, autophagic cell death, TLR4 signaling pathway, Notch signaling pathway, and mTOR signaling pathway. These pathways are thought to be closely associated with NEC. Furthermore, a lncRNA-miRNA interaction network was constructed, and four of the novel, differentially expressed lncRNAs with large changes were randomly verified using quantitative polymerase chain reaction (qPCR). The GO and KEGG pathway analysis of these four lncRNAs showed that they were associated with the negative regulation of TLR4 signaling pathway and Notch signaling pathway. In conclusion, our study revealed that these differentially expressed lncRNAs may participate in the development of NEC via interactions with miRNAs and may serve as possible biomarkers and target genes in NEC.

Human \u03b2-defensin-3 reduces excessive autophagy in intestinal epithelial cells and in experimental necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a leading cause of mortality in preterm newborns. Intestinal barrier dysfunction is one key event in NEC pathogenesis. Human β-defensin-3 (hBD3), one member of cationic host defence peptides, was reported to reduce the development of necrotizing enterocolitis in a neonatal rat model. And autophagy was induced in the intestine of human and animals with NEC. We hypothesized that regulation of autophagy might play a critical role in hBD3-mediated protection against NEC injury. Autophagy activity was evaluated both in intestinal epithelial cells and in NEC models. Newborn Sprague-Dawley rats were divided randomly into four groups: Control + NS, Control + rapamycin, NEC + NS, and NEC + hBD3. Body weight, histological score, survival time, enterocyte migration and mucosal barrier were recorded. Our results showed that hBD3 pretreatment could effectively inhibit autophagy activity in cultured IEC-6 and Caco2 enterocytes, and CXCR4 might be involved in hBD3-mediated autophagy suppression. Moreover, hBD3-induced inhibition of autophagy significantly promoted the intestinal epithelial cell migration by wound healing assay and transwell migration assay. In the rat model of NEC, hBD3 could noticeably reduce the expression of autophagy-activated proteins, down-regulate the expression of inflammatory mediators, and promote the mucosal integrity. Our data suggest an additional role of hBD3-mediated protection against intestinal mucosal injury: inhibition of over-activated autophagy in enterocytes.