Necrotizing Enterocolitis In Mice Research Articles

Expression and Role of PD-L1 in a Mouse Model of Necrotizing Enterocolitis

To investigate the expression and role of programmed death ligand-1 (PD-L1) in a mouse model of necrotizing enterocolitis (NEC). A total of 20 wild-type C57 BL/6 J mice were randomly assigned to the control and the model groups. Mice in the control group were breastfed, while mice in the model group were given lipopolysaccharide, formula feeding, hypoxia, and cold stimulation for NEC induction. Then, the intestines of the mice were collected in order to assess the pathological changes through HE staining, to examine PD-L1 expression and localization with immunofluorescence co-localization, and to evaluate intestinal PD-L1 expression with Western blot. Peripheral blood was collected for flow cytometry to examine leukocyte subpopulations and their PD-L1 expression. On the other hand, 14 PD-L1 (+/+) mice and 14 PD-L1 (-/-) mice were randomly divided into their respective genotype control groups and model groups. The same induction method as was already mentioned was adopted for the model groups. The intestines of the mice were collected for HE staining to evaluate the pathological change and peripheral blood was collected to examine the expression of inflammatory factors. The NEC mouse model was successfully constructed. PD-L1 was widely expressed in enterocytes and inflammatory cells in the mouse intestines and in T cells, monocytes, and neutrophils in peripheral blood. The expression of PD-L1 in NEC mouse intestines increased in comparison with that of the control group. In the peripheral blood of NEC mice, the proportion of T cells and monocytes and their PD-L1 expression showed no significant changes compared with those of the control group, while the proportion of neutrophils and their PD-L1 expression increased by about 140% and 150%, respectively, in comparison with those of the control group ( P<0.05). According to the results of the PD-L1 gene mouse experiment, the control groups of PD-L1 (+/+) mice and PD-L1 (-/-) mice showed no significant difference in their intestinal conditions and serum inflammatory factor levels, while the PD-L1 (-/-) NEC mouse had worse intestinal pathological changes and increased mean pathological scores compared with those of PD-L1 (+/+) NEC mouse ( P<0.05). In addition, serum interleukin (IL)-10 in PD-L1 (-/-) NEC mouse decreased by about 44% compared with that of PD-L1 (+/+) NEC mice, and chemokine (C-X-C motif) ligand 1/IL-6/IL-1β all increased by more than 25% (all P<0.05). PD-L1 is widely expressed in inflammatory cells and enterocytes in mice. Knocking out PD-L1 aggravates the degree of NEC inflammation and intestinal pathological changes. PD-L1 plays a protective role by reducing inflammation in the pathogenesis of NEC, the mechanism of which may be related to the regulation of neutrophils/enterocytes.

Nicotinamide riboside relieves the severity of experimental necrotizing enterocolitis by regulating endothelial function via eNOS deacetylation

BackgroundNicotinamide adenine dinucleotide (NAD+) is involved in regulating oxidative stress. Although NAD+ is associated with various health issues, its role in the intestinal microcirculation in necrotizing enterocolitis (NEC) remains to be confirmed. In the current study, we explored whether nicotinamide riboside (NR), a natural NAD + precursor, ameliorates the severity of NEC through endothelial nitric oxide synthase(eNOS) signaling. MethodsA mouse experimental NEC model was induced by formula gavage and hypoxia in full-term mouse pups. Intestinal endothelial cells (MIMECs) were isolated and subjected to stress using tumor necrosis factor (TNF)-α. NR was administered to assess the intestinal microcirculation and lipid peroxidation levels and to explore the involved signaling pathways. ResultsNAD + levels were reduced after induction of NEC stress, which was associated with intestinal injury. NR administration promoted NAD + levels, attenuated oxidative stress and relieved the symptoms of experimental NEC, which were relevant to increased intestinal microcirculatory perfusion through the sirtuin (SIRT) 1 pathway in experimental NEC mice. However, this improvement was not found in eNOS-knockout mice. Consistently, MIMECs exposed to TNFα showed decreased SIRT1 activity associated with increased eNOS acetylation, which could bring about endothelial dysfunction due to limited nitric oxide production. NR administration increased the NAD + content and repressed the production of reactive oxygen species (ROS) in MIMECs under TNFα stress. NR also promoted SIRT1 activity and accordingly suppressed the eNOS acetylation levels under TNFα stress. ConclusionThe current data indicate that NR administration improves the survival of experimental NEC mice via SIRT1-associated eNOS acetylation/deacetylation modulation, which is implicated in endothelial dysfunction. Although NR is commonly found in the human diet, it may also be a promising strategy for NEC treatment because of its pathogenic association with NEC.

Melatonin Alleviates Neonatal Necrotizing Enterocolitis by Repressing the Activation of the NLRP3 Inflammasome.

Objective Necrotizing enterocolitis (NEC) is one of the commonest gastrointestinal critical diseases in newborns. Several researches have proven the efficacy of melatonin (MEL) on NEC, but the latent mechanisms were ambiguous. We designed the current research to evaluate the function and mechanism of MEL on NEC in a neonatal mouse model. Methods The newborn mice were subjected to formula milk containing LPS and hypoxia to establish a NEC model and also intraperitoneally injected with MEL. During the experiment, all mice were closely monitored and weighed. The effect of MEL on the histopathological injury of the terminal ileum tissues, inflammation, and oxidative stress of serum in NEC mice was examined by hematoxylin-eosin (H&E) staining and ELISA. The effect of MEL on the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome was assessed via quantitative real-time PCR and Western blot. Results MEL intensified the survival rate and body weight in NEC mice. The H&E staining illustrated that MEL improved the histopathological injury in NEC mice. Moreover, MEL repressed the IL-1β, TNF-α, and MDA levels of serum and enhanced the SOD and GSH-Px levels of serum in NEC mice. We also discovered that MEL attenuated the mRNA and protein levels of NLRP3, Toll-like Receptor 4 (TLR4), NF-κB, and caspase-1 of the terminal ileum tissues in NEC mice. Conclusion Our research illuminated that MEL attenuated the severity of NEC via weakening the activation of the NLRP3 inflammasome.

Remote ischemic conditioning causes CD4 T cells shift towards reduced cell-mediated inflammation.

Necrotizing enterocolitis (NEC) is a gastrointestinal disease in neonates that is associated with immune-mediated intestinal inflammation. Remote ischemic conditioning (RIC) applied to a limb has been shown to be protective against experimental NEC. In this study, we explore the immune cell-mediated response involved in NEC and the immunomodulatory effects of RIC in an experimental mouse model of the disease. NEC was induced in C57BL/6 mice (ethical approval #58119) pups on postnatal day5 (p5) using gavage hyperosmolar formula, lipopolysaccharide, and hypoxia. RIC consisted of 4 cycles of 5min ischemia followed by 5min reperfusion of the right hindlimb during NEC induction on p6 and p8. Breastfed mice were used as control. The mice were sacrificed on p9, with ileal tissue evaluated for inflammatory cytokines and by characterization of T-cell populations. NEC mice had increased number of CD4+ cells indicating an accumulation of T-cells in the mesenchyme of the NEC ileum. Compared to control, NEC pups had upregulated expression pro-inflammatory cytokines (GATA3, IFNγ, IL1β, IL6, IL17, IL22, and TNFα) and reduced anti-inflammatory cytokine (TGFβ). In NEC, there was also a shift in the balance of Treg/Th17 cells towards Th17. Compared to NEC alone, RIC during the course of NEC resulted in reduction of pro-inflammatory cytokines (GATA3, IFNγ, IL1β, IL6, IL17, IL22, and TNFα), increase in anti-inflammatory cytokine TGFβ and concomitant shift back of Th17 cells towards Treg cells. In experimental NEC, remote ischemic conditioning reduces the production of pro-inflammatory markers and increases the production of anti-inflammatory markers. In addition, during NEC, RIC reverses the imbalance of Treg/Th17 providing support for its effect on cell-mediated inflammation. RIC is a non-invasive physical maneuver that can have a significant beneficial effect in reducing the inflammation seen in NEC.

Human Breast Milk-Derived Exosomal miR-148a-3p Protects Against Necrotizing Enterocolitis by Regulating p53 and Sirtuin 1.

Necrotizing enterocolitis (NEC) is a gastrointestinal disease that results in the exaggerated intestinal inflammation and injury. Human breast milk-derived exosome (BMEXO) has been reported to relieve NEC, which is closely related to the contained microRNAs (miRNAs). However, which miRNA and whether its synthesized mimic can replace the protection of BMEXO remains unclear. We established a NEC mouse model, and miRNA sequencing was performed to determine the miRNA profiling in BMEXO. The downstream target of miRNA was then confirmed by dual-luciferase reporter assay. Finally, we explored the protective effect of a single miRNA agomir on NEC and its downstream mechanisms. The results revealed that BMEXO treatment exerts a significant protective effect on NEC mice, including inhibiting inflammation and improving intercellular tight junctions. Additionally, as the most abundant miRNA in BMEXO, miR-148a-3p directly targets Tp53 on its 3' untranslated region (3' UTR). miR-148a-3p mimic treatment significantly reduces p53 expression and upregulates sirtuin 1 (SIRT1) level in the lipopolysaccharide (LPS)-treated intestinal epithelial IEC6 cells. In addition, decreased nuclear translocation of nuclear factor-κB (NF-κB) and cell apoptosis were observed by miR-148a-3p mimic. Also, delivery of miR-148a-3p agomir in vivo exerts a similar protective role on NEC as BMEXO treatment, accompanied by changes in p53 and SIRT1. Finally, the abolition of the protection of miR-148a-3p agomir on NEC was observed in a Sirt1-deficient (Sirt1+/-) mouse. Collectively, our present study demonstrated that the miR-148a-3p/p53/SIRT1 axis has a considerable protective effect on NEC, and the agomir therapy provides a new treatment strategy for NEC.

Ontogeny of ROR\u03b3t+ cells in the intestine of newborns and its role in the development of experimental necrotizing enterocolitis

BackgroundNeonates possess an immature and plastic immune system, which is a major cause of some diseases in newborns. Necrotizing enterocolitis (NEC) is a severe and devastating intestinal disease that typically affects premature infants. However, the development of intestinal immune cells in neonates and their roles in the pathological process of NEC have not been elucidated.ResultsWe examined the ontogeny of intestinal lamina propria lymphocytes in the early life of mice and found a high percentage of RORγt+ cells (containing inflammatory Th17 and ILC3 populations) during the first week of life. Importantly, the proportion of RORγt+ cells of intestinal lamina propria further increased in both NEC mice and patients tissue than the control. Furthermore, the application of GSK805, a specific antagonist of RORγt, inhibited IL-17A release and ameliorated NEC severity.ConclusionsOur data reveal the high proportion of RORγt+ cells in newborn mice may directly contribute to the development of NEC.

Sodium Butyrate Alleviates Intestinal Inflammation in Mice with Necrotizing Enterocolitis.

Objective To determine the role of sodium butyrate in intestinal inflammation via regulation of high-mobility group box-1 (HMGB1), we analyzed the potential mechanism in necrotizing enterocolitis (NEC) in a neonatal mouse model. Methods A NEC model was created with hypoxia and cold exposure and artificial overfeeding. C57BL/6 neonatal mice were randomized into three groups: the control, untreated NEC, and sodium butyrate (150 mM)-pretreated NEC groups. Pathological variations in ileocecal intestinal tissue were observed by HE staining and scored in a double-blind manner. The mRNA expression levels of HMGB1, Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), and inflammatory cytokines in intestinal tissues were determined by quantitative real-time PCR. The protein levels of HMGB1 and associated cytokines in intestinal tissues were evaluated using ELISA. The relative protein expression levels of TLR4 and NF-κB in intestinal tissues were quantified by western blot. Results Sodium butyrate administration improved the body weight and survival rate of NEC mice; relieved intestinal pathological injury; reduced the intestinal expression of HMGB1, TLR4, NF-κB, interleukin- (IL-) 1β, IL-6, IL-8, and TNF-α; and increased the intestinal expression of IL-10 (P < 0.05). Treatment with butyrate decreased the proportion of opportunistic Clostridium_sensu_stricto_1 and Enterococcus and increased the proportion of beneficial Firmicutes and Lactobacillus in the NEC model. Conclusions Sodium butyrate intervention relieves intestinal inflammation and partially corrects the disrupted intestinal flora in mice with NEC.

Toll-like receptor 4-mediated enteric glia loss is critical for the development of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants, whose pathogenesis remains incompletely understood, although activation of the Gram-negative bacterial receptor Toll-like receptor 4 (TLR4) on the intestinal epithelium plays a critical role. Patients with NEC typically display gastrointestinal dysmotility before systemic disease is manifest, suggesting that dysmotility could drive NEC development. Both intestinal motility and inflammation are governed by the enteric nervous system, a network of enteric neurons and glia. We hypothesized here that enteric glia loss in the premature intestine could lead to dysmotility, exaggerated TLR4 signaling, and NEC development. We found that intestinal motility is reduced early in NEC in mice, preceding the onset of intestinal inflammation, whereas pharmacologic restoration of intestinal motility reduced NEC severity. Ileal samples from mouse, piglet, and human NEC revealed enteric glia depletion, and glia-deficient mice (Plp1ΔDTR, Sox10ΔDTR, and BdnfΔDTR) showed increased NEC severity compared with wild-type mice. Mice lacking TLR4 on enteric glia (Sox10-Tlr4ko) did not show NEC-induced enteric glia depletion and were protected from NEC. Mechanistically, brain-derived neurotrophic factor (BDNF) from enteric glia restrained TLR4 signaling on the intestine to prevent NEC. BDNF was reduced in mouse and human NEC, and BDNF administration reduced both TLR4 signaling and NEC severity in enteric glia–deficient mice. Last, we identified an agent (J11) that enhanced enteric glial BDNF release, inhibited intestinal TLR4, restored motility, and prevented NEC in mice. Thus, enteric glia loss might contribute to NEC through intestinal dysmotility and increased TLR4 activation, suggesting enteric glia therapies for this disorder.

High-Mobility Group Box-1 Is Critical in the Pathogenesis of Mouse Experimental Necrotizing Enterocolitis.

Although high-mobility group box-1 (HMGB1) is related to the persistent intestinal inflammation in the development of necrotizing enterocolitis (NEC), the role of HMGB1 in the regulation of the intestinal microcirculation in NEC is not well understood. Therefore, we investigated the mechanism(s) by which HMGB1 regulates the generation of the following vasodilatory signals during the development of NEC: endothelial nitric oxide synthase (eNOS) and nitric oxide (NO). Experimental NEC was induced in full-term C57BL/6 mouse pups through the formula gavage and hypoxia technique. The blockade of HMGB1 was achieved with a subcutaneous injection of anti-HMGB1 antibody. Intestinal tissues and blood samples were collected at predetermined time points for the assessment of intestinal microcirculation, lipid peroxidation levels, and evaluation of eNOS activation. We found elevations in HMGB1 expression as early as 12 h after induction of NEC stress, which preceded intestinal injury. Treatment of mouse pups with HMGB1 neutralizing antibody attenuated the intestinal microvascular features and symptoms of NEC, but this improvement was not found in the eNOS knockout mice, suggesting that HMGB1 inhibition increased intestinal microcirculatory perfusion in an eNOS-dependent manner. Moreover, HMGB1 inhibition rescued NO production and eliminated O2•- production in experimental NEC mice through eNOS activation. These data indicate that excessive HMGB1 signaling is associated with the pathogenesis of NEC, suggesting that HMGB1 inhibition might be a promising strategy for NEC treatment.

Exogenous Autoinducer-2 Rescues Intestinal Dysbiosis and Intestinal Inflammation in a Neonatal Mouse Necrotizing Enterocolitis Model.

Autoinducer-2 (AI-2) is believed to be a bacterial interspecies signaling molecule that plays an important role in the regulation of the physiological behaviors of bacteria. The effect of AI-2 on the process of necrotizing enterocolitis (NEC) is unknown, and the aim of this study was to study the effect of AI-2 in a mouse NEC model. C57BL/6 mouse pups were randomly divided into three groups: the control group, the NEC group, and the NEC+AI-2 (NA) group. Exogenous AI-2 (500 nM) was added to the formula milk of the NA group. The concentrations of fecal AI-2 and flora were tested. The expression of cytokines, TLR4 and NF-κB in intestinal tissue was detected. The AI-2 level was significantly decreased in the NEC group (P<0.05). Compared with the NEC group, the intestinal injury scores, expression of TLR4, NF-kB, and proinflammatory factors (IL-1β, IL-6, IL-8 and TNF-α) were reduced, and expression of anti-inflammatory factor (IL-10) was increased in the NA group mice (P<0.05). At the phylum level, the Proteobacteria abundance in the NA group was significantly increased, while the Bacteroidota abundance in the control group was significantly increased (P<0.05). At the genus level, Helicobacter and Clostridium_sensu_stricto_1 exhibited significantly greater abundance in the NEC group than in the other two groups, while Lactobacillus had the opposite trend (P<0.05). In addition, the abundances of Klebsiella, Rodentibacter and Enterococcus were significantly higher in the NA group than in the NEC and control groups (P < 0.05). Exogenous AI-2 partially reverses flora disorder and decreases inflammation in an NEC mouse model.

Neonatal Intestinal Macrophages Promote Microvascular Development through an IGF‐1‐dependent Mechanism and Lack of Macrophage‐derived IGF‐1 Predisposes Newborn Mice to Necrotizing Enterocolitis

Defective intestinal microvascular development has been previously shown to play a role in necrotizing enterocolitis (NEC) pathogenesis. Decreased serum levels of IGF-1 in premature infants are associated with a higher risk of developing NEC and we recently found that serum and intestinal tissue IGF-1 level are decreased in a neonatal mouse NEC model. In the embryo, macrophages have been reported to promote angiogenesis. Here we hypothesize that neonatal intestinal macrophages promote endothelial cell proliferation and angiogenesis by producing IGF-1, thus protecting against NEC development. For this purpose, we first examined the role of IGF-1 on intestinal endothelial cell sprouting in vitro using IGF-1 and IGF-1R inhibitor and on intestinal endothelial cell proliferation in vivo using IGF-1R inhibitor. Secondly, using mice with IGF-1-deficient-macrophages and IGF-1-sufficient littermates, we assessed whether macrophage-derived IGF-1 plays a role in perinatal microvascular development by examining endothelial cell proliferation in vivo and endothelial cell sprouting in vitro in Matrigel co-cultures. Finally, we examined whether macrophage-derived IGF-1 protects against NEC. We found that, neonatal intestinal CX3CR1+ macrophages were essential for small intestinal endothelial cells to sprout in Matrigel, which process was further enhanced by exogenous IGF-1 and inhibited by the selective IGF-1R inhibitor picropodophyllin (PPP). Intestinal endothelial cell proliferation was also inhibited by PPP in vivo, to a similar extent as observed during NEC development. Furthermore, neonatal IGF-1-deficient macrophages significantly decreased pro-angiogenic activity in vitro. Mice with IGF-1-deficient-macrophages had less robust intestinal endothelial proliferation and decreased intestinal VEGF protein expression in vivo. Lastly, pups with IGF-1-deficient macrophages had increased incidence of severe NEC-like intestinal injury and mortality. In conclusion, neonatal intestinal macrophages are critical for promoting intestinal microvascular growth and for protecting pups from NEC development via IGF-1 production.

Inhibition of FLT‐1 Decreases the Incidence of Severe Necrotizing Enterocolitis in Neonatal Mice

Necrotizing enterocolitis (NEC) is a common gastrointestinal disease among premature infants. While advances in clinical management have been achieved through therapeutical modalities and surgical interventions, serious limitation in the improvement of outcomes after the diagnosis remain, largely due to the complexity of underlying biology. Our previous study suggests that maldevelopment of the intestinal microvasculature plays an important role in NEC pathogenesis and that embryonic macrophages support intestinal microvasculature development by promoting VEGF/VEGFR2 signaling and endothelial cell proliferation. We also found that monocytes express VEGFR1 which is known to trap VEGF, making it unavailable to reach VEGFR2, and that during experimental NEC, intestinal monocyte recruitment and activation mediate intestinal tissue injury and blocking monocyte NF-KB activation preserved intestinal microvascular density. However, the mechanism remains unclear. Here we hypothesize that blocking VEGFR1 protects against NEC. To test our hypothesis, littermates were injected with anti-FLT-1 antibodies (5mg/kg) or control IgG antibody (controls) and submitted one hour later to an established murine neonatal NEC model, which include inoculation with adult commensal bacteria, hypoxia, cold stress and formula feedings. Animals were euthanized when presenting signs of distress or after 72h. Intestinal tissues were collected for H&E staining and intestinal injury score determination. In pups treated with anti-FLT-1 antibodies, survival was significantly improved compared to control pups (median survival of 54h vs. 36 h respectively; p≤0.05). Moreover, intestinal injury score was significantly less in pups treated with anti-FLT-1 antibodies compared to littermate controls (10/29 vs 18/30 with NEC score ≥2, χ2=3.85, p≤0.05). In conclusion, our data shows that blocking FLT-1 increases survival and protects against intestinal injury in a neonatal mouse NEC model, suggesting a role for FLT-1 in NEC. Further investigation of this pathway may lead to the identification of potential therapeutic targets.

Maternal aryl hydrocarbon receptor activation protects newborns against necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a disease of premature infants characterized by acute intestinal necrosis. Current dogma suggests that NEC develops in response to post-natal dietary and bacterial factors, and so a potential role for in utero factors in NEC remains unexplored. We now show that during pregnancy, administration of a diet rich in the aryl hydrocarbon receptor (AHR) ligand indole-3-carbinole (I3C), or of breast milk, activates AHR and prevents NEC in newborn mice by reducing Toll-like receptor 4 (TLR4) signaling in the newborn gut. Protection from NEC requires activation of AHR in the intestinal epithelium which is reduced in mouse and human NEC, and is independent of leukocyte activation. Finally, we identify an AHR ligand (“A18”) that limits TLR4 signaling in mouse and human intestine, and prevents NEC in mice when administered during pregnancy. In summary, AHR signaling is critical in NEC development, and maternally-delivered, AHR-based therapies may alleviate NEC.

Hepatic oxidative injury: role of mitochondrial dysfunction in necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a severe neonatal gastrointestinal disease that can cause damage to remote organs. Previous studies have shown that inflammatory and oxidative injury occur in the liver during NEC. Mitochondrial DNA (mtDNA) plays an important role in hepatic injuries of many other diseases. We aimed to investigate the mechanism of mitochondrial dysfunction in hepatic oxidative injury during NEC. NEC was induced in C57BL/6 mice (approval: 44032) by hypoxia, gavage feeding with hyperosmolar formula, and lipopolysaccharide administration from postnatal days 5 to 9 (n = 15). Two additional groups with hypoxia only (n = 10) and hypoxia and hyperosmolar formula (n = 13) were also examined. Breastfed pups were used as control (n = 15). Liver was harvested on postnatal day 9. Gene expressions of mtDNA markers cytochrome c oxidase subunit 3 (COX3), cytochrome b (CYTB) and NADH-ubiquinone oxidoreductase chain 1 (ND1) were measured by real-time qPCR. Mitochondrial morphology marker HSP60 and oxidative stress marker NRF2 were detected by immunofluorescence staining and compared between NEC and control. Data were presented as mean ± SD and compared using Student's t test; p < 0.05 was considered significant. Gene expression of mtDNA markers (COX3, CYTB, and ND1) were significantly decreased in the liver of NEC mice relative to control, hypoxia alone, and hypoxia with hyperosmolar formula. Immunofluorescence showed depletion of HSP60 indicating decreased mitochondria in NEC liver relative to control. Furthermore, a higher protein expression of NRF2 was observed indicating higher oxidative stress in NEC liver relative to control. Intestinal injury in experimental NEC leads to a systemic inflammatory response affecting the liver. Hepatic oxidative injury in NEC is characterized by decreased mitochondria and mtDNA depletion. This study provides insight into the mechanism of liver injury in NEC.

MiR-146a-5p Mimic Inhibits NLRP3 Inflammasome Downstream Inflammatory Factors and CLIC4 in Neonatal Necrotizing Enterocolitis

Objective: Necrotizing enterocolitis (NEC) is a gastrointestinal emergency with a severe inflammation storm, intestinal necrosis, and perforation. MicroRNA-146a-5p (miR-146a-5p) has been reported to be a valuable anti-inflammatory factor in various intestinal inflammatory disorders. However, the role of miR-146a-5p in NEC, its effects on nucleotide-binding domain and leucine-rich repeat-containing protein 3 (NLRP3) inflammasome, and its downstream inflammatory factors remain unknown. This study aimed to investigate the role of miR-146a-5p and NLRP3 inflammasome and its downstream inflammatory factors in NEC development.Methods: The expression levels of miR-146a and NLRP3 inflammasome were investigated in intestinal tissues. Next, the mechanism by which miR-146a-5p regulates NLRP3 inflammasome activation was explored in vitro in THP-1 cells. Finally, to identify the effects of miR-146a-5p on NEC in vivo, NEC mice were transinfected with miR-146a-5p overexpression adenovirus before the occurrence of NEC.Results: NLRP3 inflammasome enzymatic protein caspase-1 and its downstream inflammatory factors increased in NEC intestinal samples in both humans and mice, and miR-146a-5p expression level was increased and mainly expressed in the macrophages of the affected intestine. In vitro, only miR-146a-5p mimic inhibited NLRP3 inflammasome downstream inflammatory factors and its upstream protein chloride intracellular channel protein 4 (CLIC4) expression in cellular membrane in the THP-1 cell line, and this only occurred under mild/moderate LPS concentration. MiR-146a-5p overexpression adenovirus transfection reduced CLIC4 cellular membrane expression and inhibited NLRP3 downstream factors increasing in vivo. After the transfection of miR-146a-5p adenovirus, the survival rate of NEC mice was increased, and intestinal injury was ameliorated.Conclusion: MiR-146a-5p inhibited NLRP3 inflammasome downstream inflammatory factors and CLIC4 membrane expression in NEC. Additionally, miR-146a-5p could attenuate inflammation and intestinal injury in the NEC-affected intestine.

Neutrophil extracellular trap inhibition increases inflammation, bacteraemia and mortality in murine necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease affecting primarily premature infants. The disease is characterized by intestinal inflammation and leucocyte infiltration, often progressing to necrosis, perforation, systemic inflammatory response and death. Neutrophil extracellular traps (NETs), denoting nuclear DNA, histone and antimicrobial protein release, have been suggested to play a role in NEC. This study aimed to determine the role of NETs in NEC and explore the effect of chloramidine, a NET inhibitor, on a murine NEC‐like intestinal injury model. Blood and intestinal tissues were collected from infants diagnosed with ≥ Stage II NEC, and levels of nucleosomes and NETs, respectively, were compared with those of case‐matched controls. In mice, NEC was induced with dithizone/Klebsiella, and mice in the treatment group received 40 mg/kg chloramidine. Bacterial load, intestinal histology, plasma myeloperoxidase and cytokine levels, and immunofluorescent staining were compared with controls. Nucleosomes were significantly elevated in both human and mouse NEC plasma, whereas NET staining was only present in NEC tissue in both species. Chloramidine treatment increased systemic inflammation, bacterial load, organ injury and mortality in murine NEC. Taken together, our findings suggest that NETs are critical in the innate immune defence during NEC in preventing systemic bacteraemia.

Saccharomyces boulardii modulates necrotizing enterocolitis in neonatal mice by regulating the sirtuin1/NF‑κB pathway and the intestinal microbiota.

Exaggerated inflammatory response and gut microbial dysbiosis play a crucial role in necrotizing enterocolitis (NEC). The probiotic Saccharomyces boulardii (SB) is a yeast that has a beneficial effect on NEC; however, the association between its protective effects and the regulation of the inflammation-related sirtuin 1 (SIRT1)/nuclear factor-κB (NF-κB) signaling pathway and gut microbiota in NEC is unknown. In the present study, the NEC model was established by artificial feeding and lipopolysaccharide (LPS), hypoxia and hypothermia stimulation. Mice were divided into normal, control (artificial feeding), NEC and NEC + SB groups. Hematoxylin and eosin staining demonstrated that SB improved the pathological damage of the intestine caused by NEC in neonatal mice. Furthermore, downregulation of SIRT1 and upregulation of NF-κB expression were confirmed by immunofluorescence staining, western blotting and reverse transcription-quantitative PCR (RT-qPCR) in NEC mice. SB treatment concurrently inhibited the NEC roles on the SIRT1 and NF-κB pathway at both the protein and mRNA levels. Deletion of SIRT1 [SIRT1 knockout (KO)] in the intestine abolished all the effects of SB in NEC mice, including protection of pathological damage and inhibition of the SIRT1/NF-κB pathway activation. The abundance of gut microbial composition, as determined by RT-qPCR, was significantly decreased in the control group compared with the normal group. A further decrease in microbiota abundance was observed in the NEC group, and SB administration significantly improved the enrichment of gut microbiota in neonatal mice with NEC. As anticipated, the increased abundance of gut microbiota modulated by SB was markedly reduced in SIRT1KO NEC mice. The present study revealed that the protective role of SB on NEC was associated with the SIRT1/NF-κB pathway and gut microbiota regulation.

The human milk oligosaccharides 2'-fucosyllactose and 6'-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling.

Objective:Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2’-Fucosyllactose (2’-FL) and 6’-Sialyllactose (6’-SL), can reduce NEC through inhibition of TLR4 signaling.Design:NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2’-FL, 6’-SL or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, human tissue explants, and via in silico modeling.Results:Supplementation of infant formula with either 2’-FL and/or 6’-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2’-FL and 6’-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2’-FL and 6’-SL, but not lactose, to dock into the binding pocket of the TLR4-MD2 complex, explaining their ability to inhibit TLR4 signaling.Conclusion:2’-FL and 6’-SL, but not lactose, prevent NEC in mice and piglet models, and attenuate NEC-inflammation in human ileum, in part through TLR4 inhibition.

Beneficial effects of butyrate in intestinal injury

PurposeButyrate is a short-chain fatty acid produced in the intestine. It is controversial whether butyrate is protective or destructive for the intestinal epithelium in the development of diseases like necrotizing enterocolitis (NEC), and its mechanism of action remains unclear. We aimed to determine the effect of butyrate on the intestinal epithelium by studying its effects on intestinal epithelial cells (IEC-18) exposed to injury and in vivo by investigating the effects on the intestine in an experimental model of NEC. MethodsA) In vitro study: Butyrate was given to normal IEC-18 to determine the dose triggering injury. Based on above results, low dose butyrate (1 mM) was given to H2O2-injured cells to determine its effect against inflammation. B) In vivo study: NEC was induced by hypoxia and gavage feeding between postnatal day P5 and P9 (n = 8). Breastfed mice were used as control (n = 7). Butyrate (150 mM) was administered by enema on P6 in NEC (n = 6). Distal ileum was harvested on P9. ResultsHigh dose (16 mM) butyrate upregulated inflammatory marker IL-6, while low dose butyrate protected cells from injury by reducing IL-6 expression. Similarly, compared with NEC alone, NEC mice who received butyrate had reduced intestinal damage, reduced IL-6 and NF-ĸB expression, and increased intestinal tight junction marker Claudin-7. ConclusionButyrate has opposite effects depending on the dose administered. Butyrate can protect cells from H2O2-induced injury and can in vivo protect the intestine from NEC. This beneficial effect is because of downregulation of inflammation and enhancement of intestinal barrier.

Calcium/calmodulin-dependent protein kinase IV signaling pathway is upregulated in experimental necrotizing enterocolitis.

Activation of calcium/calmodulin-dependent protein kinase IV (CaMKIV) has been shown to increase intestinal injury and inhibit epithelial cell proliferation in dextran sulfate sodium (DSS)-induced colitis mice. However, the role of CaMKIV in necrotizing enterocolitis (NEC) is unknown. We aimed to study the expression and activation of CaMKIV in experimental NEC. Following ethical approval, NEC (n = 10) was induced in C57BL/6 mouse pups by hypoxia, gavage hyperosmolar formula feeding and lipopolysaccharide from postnatal days P5 to 9. Breastfed pups served as control (n = 10). Mouse pups were sacrificed on P9 and the terminal ileum was harvested. Gene NEC injury was scored blindly by three independent investigators. CaMKIV, CREM and IL17 gene expression, and CaMKIV and pCaMKIV protein expression were assessed. The data were compared using Mann-Whitney U test. P < 0.05 was considered significant. Intestinal injury was induced in the NEC mice and confirmed by histological scoring and inflammatory cytokine IL6. CaMKIV and its downstream target genes of CREM and IL17 were significantly elevated in NEC mice relative to control. Similarly, phosphorylated-CaMKIV (pCaMKIV), the active form of CaMKIV, was more notably expressed in the NEC ileal tissue relative to control ileal tissue. Elevated pCaMKIV protein expression was also confirmed by western blot. CaMKIV expression and activation are upregulated in experimental NEC suggesting a potential contributing factor in the pathogenesis of NEC.