Necrotizing Enterocolitis In Mice Research Articles

RNF31-mediated IKKα ubiquitination aggravates inflammation and intestinal injury through regulating NF-κB activation in human and mouse neonates

AimsNeonatal necrotizing enterocolitis (NEC) is a leading cause of intestine inflammatory disease, and macrophage is significantly activated during NEC development. Posttranslational modifications (PTMs) of proteins, particularly ubiquitination, play critical roles in immune response. This study aimed to investigate the effects of ubiquitin-modified proteins on macrophage activation and NEC, and discover novel NEC-related inflammatory proteins. Materials and methodsProteomic and ubiquitin proteomic analyses of intestinal macrophages in NEC/healthy mouse pups were carried out. In vitro macrophage inflammation model and in vivo NEC mouse model, as well as clinical human samples were used for further verification the inhibitor of nuclear factor-κB kinase α (IKKα) ubiquitination on NEC development through Western blot, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry. Key findingsWe report here that IKKα was a new ubiquitin-modified protein during NEC through ubiquitin proteomics, and RING finger protein 31 (RNF31) acted as an E3 ligase to be involved in IKKα degradation. Inhibition of IKKα ubiquitination and degradation with siRNF31 or proteasome inhibitor decreased nuclear factor-κB (NF-κB) activation, thereby decreasing the expression of pro-inflammatory factors and M1 macrophage polarization, resulting in reliving the severity of NEC. SignificanceOur study suggests the activation of RNF31-IKKα-NF-κB axis triggering NEC development and suppressing RNF31-mediated IKKα degradation may be therapeutic strategies to be developed for NEC treatment.

Umbilical cord mesenchymal stem cell exosomes alleviate necrotizing enterocolitis in neonatal mice by regulating intestinal epithelial cells autophagy

BACKGROUND Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease that affects premature infants. Although mounting evidence supports the therapeutic effect of exosomes on NEC, the underlying mechanisms remain unclear. AIM To investigate the mechanisms underlying the regulation of inflammatory response and intestinal barrier function by umbilical cord mesenchymal stem cell (UCMSCs) exosomes, as well as their potential in alleviating NEC in neonatal mice. METHODS NEC was induced in 5-d-old C57BL/6 pups through hypoxia and gavage feeding of formula containing lipopolysaccharide (LPS), after which the mice received human UCMSC exosomes (hUCMSC-exos). The control mice were allowed to breastfeed with their dams. Ileal tissues were collected from the mice and analyzed by histopathology and immunoblotting. Colon tissues were collected from NEC neonates and analyzed by immunofluorescence. Molecular biology and cell culture approaches were employed to study the related mechanisms in intestinal epithelial cells. RESULTS We found that autophagy is overactivated in intestinal epithelial cells during NEC, resulting in reduced expression of tight junction proteins and an increased inflammatory response. The ability of hUCMSC-exos to ameliorate NEC in a mouse model was dependent on decreased intestinal autophagy. We also showed that hUCMSC-exos alleviate the inflammatory response and increase migration ability in intestinal epithelial cells induced by LPS. CONCLUSION These results contribute to a better understanding of the protective mechanisms of hUCMSC-exos against NEC and provide a new theoretical and experimental foundation for NEC treatment. These findings also enhance our understanding of the role of the autophagy mechanism in NEC, offering potential avenues for identifying new therapeutic targets.

Umbilical cord mesenchymal stem cell exosomes alleviate necrotizing enterocolitis in neonatal mice by regulating intestinal epithelial cells autophagy

BACKGROUND Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease that affects premature infants. Although mounting evidence supports the therapeutic effect of exosomes on NEC, the underlying mechanisms remain unclear. AIM To investigate the mechanisms underlying the regulation of inflammatory response and intestinal barrier function by umbilical cord mesenchymal stem cell (UCMSCs) exosomes, as well as their potential in alleviating NEC in neonatal mice. METHODS NEC was induced in 5-d-old C57BL/6 pups through hypoxia and gavage feeding of formula containing lipopolysaccharide (LPS), after which the mice received human UCMSC exosomes (hUCMSC-exos). The control mice were allowed to breastfeed with their dams. Ileal tissues were collected from the mice and analyzed by histopathology and immunoblotting. Colon tissues were collected from NEC neonates and analyzed by immunofluorescence. Molecular biology and cell culture approaches were employed to study the related mechanisms in intestinal epithelial cells. RESULTS We found that autophagy is overactivated in intestinal epithelial cells during NEC, resulting in reduced expression of tight junction proteins and an increased inflammatory response. The ability of hUCMSC-exos to ameliorate NEC in a mouse model was dependent on decreased intestinal autophagy. We also showed that hUCMSC-exos alleviate the inflammatory response and increase migration ability in intestinal epithelial cells induced by LPS. CONCLUSION These results contribute to a better understanding of the protective mechanisms of hUCMSC-exos against NEC and provide a new theoretical and experimental foundation for NEC treatment. These findings also enhance our understanding of the role of the autophagy mechanism in NEC, offering potential avenues for identifying new therapeutic targets.

MiR-375-3p targets YWHAB to attenuate intestine injury in neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a significant contributor to neonatal mortality. This study aimed to investigate the role of high levels of miR-375-3p in breast milk in the development of NEC and elucidate its mechanism. Differential expression of miR-375-3p in the intestines of breast-fed and formula-fed mice was confirmed using real-time polymerase chain reaction (RT-PCR). NEC mice models were established, and intestinal injury was assessed using HE staining. RT-PCR and Western blot were conducted to examine the expression of miR-375-3p, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein β (YWHAB), as well as the inflammatory in IEC-6 cells, and intestinal tissues obtained from NEC mice and patients. Flow cytometry and cell counting kit-8 (CCK-8) were employed to elucidate the impact of miR-375-3p and YWHAB on cell apoptosis and proliferation. Breastfeeding increases miR-375-3p expression in the intestines. The expression of miR-375-3p in NEC intestinal tissues exhibited a significant decrease compared to the healthy group. Additionally, the expression of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) was higher in the NEC group compared to the control group. Down-regulation of miR-375-3p inhibited IEC-6 cell proliferation, increased apoptosis, and elevated secretion of inflammatory factors. Bioinformatics revealed that YWHAB may be a target of miR-375-3p. RT-PCR and Western blot indicated a down-regulation of YWHAB expression in intestines of NEC patients and mice. Furthermore, YWHAB was found to be positively connected with miR-375-3p. Knockdown miR-375-3p down-regulated YWHAB expression in cells. Inhibition of YWHAB exhibited similar effects to miR-375-3p in IEC-6 cells. YWHAB plasmid partially reverse cellular functional impairment induced by miR-375-3p knockdown. Breastfeeding elevated miR-375-3p expression in intestines in neonatal mice. MiR-375-3p leads to a decrease in apoptosis of intestinal epithelial cells, an increase in cell proliferation, and a concomitant reduction in the expression of inflammatory factors partly through targeting YWHAB.

Recombinant IGF-1/BP3 protects against intestinal injury in a neonatal mouse NEC model.

Recombinant human IGF-1/binding protein-3 (rhIGF-1/BP3) is currently being tested in phase II clinical trials in premature infants to prevent bronchopulmonary dysplasia, but its impact on the neonatal intestine remains unclear. The aim of this study was to determine whether rhIGF-1/BP3 protects against necrotizing enterocolitis (NEC) in mice and to investigate the mechanisms involved. Neonatal mice were dam fed or injected intraperitoneally with rhIGF-1/BP3 (or vehicle) and submitted to an experimental NEC model. Serum IGF-1 was assessed by ELISA and intestinal vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expression by Western blot. Intestinal endothelial cell proliferation, and enterocyte proliferation and migration were examined by immunofluorescence. Pup survival and histological intestinal injury were determined. In pups exposed to experimental NEC, serum IBP3-bound IGF-1 level was decreased. Exogenous rhIGF-1/BP3 preserved VEGF and VEGFR2 protein expression, decreased vascular permeability, and preserved endothelial cell proliferation in the small intestine. Furthermore, rhIGF-1/BP3 promoted enterocyte proliferation and migration, which effects were attenuated by inhibiting VEGFR2 signaling, decreased enterocyte apoptosis and decreased systemic and intestinal inflammation. rhIGF-1/BP3 improved survival and reduced the incidence of severe intestinal injury in experimental NEC. Exogenous rhIGF-1/BP3 protects neonatal mice against experimental NEC via multiple mechanisms. Exogenous rhIGF-1/BP3 preserves intestinal microvascular development and integrity, promotes enterocyte proliferation and migration, decreases local and systemic inflammation, and protects neonatal mice against NEC. The article adds pre-clinical evidence of a protective role for rhIGF-1/BP3 on the premature gut. It provides evidence supporting the use of rhIGF1/BP3 in premature neonates to protect against NEC.

Melatonin alleviates necrotizing enterocolitis by reducing bile acid levels through the SIRT1/FXR signalling axis

Bile acids (BAs) have increasingly been implicated in the onset and progression of necrotizing enterocolitis (NEC); multiple findings have demonstrated their ability to induce damage to the intestinal epithelium, thereby exacerbating disease severity. Although we previously showed that melatonin was able to treat NEC by correcting the Treg/Th17 imbalance, the modulatory effect of melatonin on BAs remains unclear. In this study, we conducted transcriptome analysis on intestinal tissues from patients with NEC and validated these findings. Subsequently, we treated mice with melatonin alone or in combination with an agonist/inhibitor of Sirtuin 1 (SIRT1) to assess faecal and serum BA levels, the expression levels of BA transporters and regulators, and the extent of intestinal injury. Our transcriptome results indicated dysregulation of BA metabolism and abnormal expression of BA transporters in patients with NEC, which were also observed in our NEC mouse model. Furthermore, exogenous BAs were found to aggravate NEC severity in mice. Notably, melatonin effectively restored the aberrant expression of BA transporters, such as apical membrane sodium-dependent bile acid transporters (ASBT), ileal bile acid-binding protein (IBABP), and organic solute transporter-alpha (OST-α), by upregulating SIRT1 expression while reducing farnesoid X receptor (FXR) acetylation, consequently leading to decreased serum and faecal BA levels and mitigated NEC severity. Thus, we propose a potential mechanism through which melatonin reduces BA levels via the SIRT1/FXR signalling axis in an NEC mouse model. Collectively, these results highlight that melatonin holds promise for reducing BA levels and represents a promising therapeutic strategy for treating NEC.

Butyrate suppresses experimental necrotizing enterocolitis-induced brain injury in mice.

Necrotizing enterocolitis (NEC) is a devastating disease in premature infants, and 50% of infants with surgical NEC develop neurodevelopmental defects. The mechanisms by which NEC-induced cytokine release and activation of inflammatory cells in the brain mediate neuronal injury, and whether enteral immunotherapy attenuates NEC-associated brain injury remain understudied. Based on our prior work, which demonstrated that experimental NEC-like intestinal injury is attenuated by the short-chain fatty acid, butyrate, in this study, we hypothesize that NEC-induced brain injury would be suppressed by enteral butyrate supplementation. A standardized NEC mouse model [enteral formula feeding, lipopolysaccharide (LPS), and hypoxia] was used. Mice were randomized into the following groups: control, NEC, butyrate pretreated NEC, and butyrate control. NEC scoring (1-4 with 4 representing severe injury) was performed on ileal sections using a validated scoring system. Intestinal and brain lysates were used to assess inflammation, proinflammatory signaling, and apoptosis. NEC-induced intestinal injury was attenuated by butyrate supplementation. NEC-induced microglial activation in the cerebral cortex and hippocampus was suppressed with butyrate. NEC increased the number of activated microglial cells but decreased the number of oligodendrocytes. Butyrate pretreatment attenuated these changes. Increased activation of proinflammatory Toll-like receptor signaling, cytokine expression, and induction of GFAP and IBA1 in the cerebral cortex observed with NEC was suppressed with butyrate. Experimental NEC induced inflammation and activation of microglia in several regions of the brain, most prominently in the cortex. NEC-induced neuroinflammation was suppressed with butyrate pretreatment. The addition of short-chain fatty acids to diet may be used to attenuate NEC-induced intestinal injury and neuroinflammation in preterm infants.

Clostridium butyricum and Clostridium tyrobutyricum: angel or devil for necrotizing enterocolitis?

This study sheds light on that treatment with Clostridium tyrobutyricum but not Clostridium butyricum is entitled to protect against necrotizing enterocolitis (NEC) development potentially. The mechanisms behind the opposite effect on NEC may result in different modulation on the level of Akkermansia muciniphila, which is deeply associated with intestinal homoeostasis. Briefly, through improving the abundance of A. muciniphila to alleviate intestinal inflammation and enhance intestinal barrier integrity, C. tyrobutyricum supplement may become a promising therapy for NEC.

The Protective Effect of Sulforaphane on ER-induced Apoptosis and Inflammation in Necrotizing Enterocolitis Mice.

Necrotizing enterocolitis (NEC) is a neonatal intestinal necrotizing disease caused by various factors in newborns. Sulforaphane (SFN) has a strong anti-inflammatory ability and a certain protective effect on intestinal diseases. NEC is a common developed gastrointestinal exigency in an untimely baby. SFN is a naturally originated isothiocyanate that has beneficial effects on the intestinal system.The purpose of this study is to study the protective effect of SFN on endoplasmic reticulum stress (ERS)-related NEC. The newborn mice were randomly divided into control (n = 15), NEC (n = 20), and NEC+SFN (n = 18) groups. Mice in NEC and SFN+NEC groups were injected with 0.1 μl normal saline or 20 mg/kg/d SFN, respectively. After that, the weight and survival of the mice were recorded every day. Then the mice were sacrificed after 96 h of modeling; ileum tissue and blood samples were collected for qPCR, Western blot, ELISA, HE staining, TUNEL staining, and immunohistochemistry assays. SFN significantly inhibited the mRNA expression of BIP, CHOP, IL-1β and IL-6, and protein expression of Bax, Caspase-3, Caspase-9 and CHOP, and promoted the expression of Bcl-2 in ER-induced NEC mice intestinal tissues (P<0.01). Meanwhile, SFN could suppress the serum levels of IL-8, IL-10, IL-6, TNF-α, and IL-1β, and positive expression of TLR4 and NF-κB (P<0.01), and promote the serum levels of IL-10. HE staining showed that SFN alleviated the NEC intestinal tissue injury, and TUNNEL staining showed that SFN could reduce the rate of NEC apoptotic cells (P<0.01). Moreover, SFN treatment improved the body weight and survival rate in NEC mice. SFN could effectively protect against ERS-induced inflammation and apoptosis in NEC mice.

Remote Ischemic Conditioning Relieves Necrotizing Enterocolitis Through Regulation of Redox and Inflammation.

In neonates, necrotizing enterocolitis (NEC) is a serious condition involving oxidative stress and inflammation. Remote ischemic conditioning (RIC) is a potentially useful technique to protect distant organs from the damage induced by ischemia. RIC has been verified as effective to protect against NEC; however, its mechanism is unclear. This study aimed to assess the mechanism and efficacy of RIC to treat experimental NEC in mice. Between postnatal day (P) 5 and P9, we induced NEC in C57BL/6 mice and Grx1-/- mice. Intermittent occlusion of the blood flow to the right hind limb for 4 cycles of 5 min ischemia followed by 5 min reperfusion during NEC induction on P6 and P8 was used to apply RIC. We sacrificed the mice on p9 and evaluated oxidative stress, inflammatory cytokines, proliferation, apoptosis, and PI3K/Akt/mTOR signal pathway in mice ileal tissue. RIC decreased intestinal injury and prolonged survival in NEC pups. RIC significantly inhibited inflammatory, attenuated oxidative stress, reduced apoptosis, promoted proliferation, and activated PI3K/Akt/mTOR in vivo. RIC activates the PI3K/Akt/mTOR signaling pathway to control oxidative stress and inflammation. RIC might provide a new therapeutic strategy for NEC.

Knockdown of PHLDA1 Alleviates Necrotizing Enterocolitis by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis Through Enhancing Nrf2 Signaling

ABSTRACT Objective Pleckstrin homology-like domain family A member 1 (PHLDA1) is involved in the progression of intestine-related diseases, but its role and related mechanisms in Necrotizing enterocolitis (NEC) are unclear. The aim of this study was to better understand the function of PHLDA1 in NEC and the underlying mechanisms. Methods A neonatal mouse model of NEC was established by hypoxic hypothermia, and sh-PHLDA1 was transfected into mice to observe the mortality of each group within 4 days. The levels of IL-1β, IL-6, IL-18 and TNF-α were measured by PCR and ELISA. ROS, MDA, SOD, and GSH-Px levels were detected by Dihydroethidium (DHE) method and kit; expression of pyroptosis-related factors including NLRP3, ASC, cleaved-caspase1, GSDMD-N, IL-1β, IL-18, and Nrf2 were detected by western-blot; mechanistically, the effects of transfection of sh-PHLDA1 and ML385 (Nrf2 inhibitor) were investigated, and the expression of pyroptosis-related factors was detected again. Results PHLDA1 was highly expressed in the intestinal tissues of NEC mice, and transfection of sh-PHLDA1 improved the survival rate, alleviated intestinal lesions, improved intestinal inflammation, oxidative stress and cellular scorching in NEC. In addition, sh-PHLDA1 was able to inhibit NLRP3 activation and pyroptosis by activating Nrf2. Conclusion Knockdown of PHLDA1 attenuated necrotizing small intestinal colitis by enhancing Nrf2 expression to inhibit NLRP3 inflammasome activation and pyroptosis.

Protective Effect of the SIRT1-Mediated NF-κB Signaling Pathway against Necrotizing Enterocolitis in Neonatal Mice.

To discover the mechanism of the sirtuin 1 (SIRT1)-mediated nuclear factor-κB (NF-κB) pathway in the protection against necrotizing enterocolitis (NEC) in neonatal mice. Neonatal mice were treated with EX527 (an inhibitor of SIRT1) and/or pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB). The survival rate of the mice was recorded. Hematoxylin and eosin (HE) staining was performed to observe the pathological changes in the intestines. Furthermore, western blotting, enzyme-linked immunosorbent assay, and real-time quantitative polymerase chain reaction were conducted to measure the protein and gene expression, while corresponding kits were used to detect the levels of oxidative stress indicators. PDTC increased the survival rate of NEC mice. When compared with the NEC+ EX527 + PDTC group, the histological NEC score was higher in the NEC + EX527 group but lower in the NEC + PDTC group. SIRT1 expression in the intestines of NEC mice was downregulated, with an increase in p65 nuclear translocation. Additionally, malondialdehyde increased and glutathione peroxidase decreased in the intestines of NEC mice, with the upregulation of interleukin (IL)-6, IL-1β, and tumor necrosis factor-α, as well as the downregulation of ZO-1, occludin, and claudin-4 in the intestines. However, the above changes could be improved by PDTC, which could be further reversed by EX527. SIRT1 can mitigate inflammation and the oxidative stress response and improve intestinal permeability by mediating the NF-κB pathway, playing an important role in the alleviation of NEC.

Protective Effects of Lactobacillus reuteri on Intestinal Barrier Function in a Mouse Model of Neonatal Necrotizing Enterocolitis.

The intestinal mucosal and immune barriers play considerable roles in the pathogenesis of necrotizing enterocolitis (NEC). The present research was designed to assess the protective effects of Lactobacillus reuteri (LR) DSM 17938 (LR 17938) on the intestinal barriers and its beneficial effects on inflammation in a neonatal mouse model of NEC. Overall, 7-day-old 75 C57BL/6 neonatal mice were separated into three groups (n = 25) as follows: (1) control, (2) NEC, and (3) NEC + LR17938 (LR group). NEC mice were administered a hypertonic feeding formula and subjected to asphyxia and hypothermia. Hematoxylin and eosin (HE) staining and pathological scores were used to assess the pathological changes in the intestine. Oxidative stress was evaluated based on the levels of superoxide dismutase (SOD) and malondialdehyde (MDA). Tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels were detected to assess inflammation. Gut permeability levels, bacterial translocation, and the levels of secretory idioglobulin A (sIgA), β-defensin, and tight junction (TJ) proteins were detected to evaluate gut mucosal and immune barrier function, and gut microbial diversity was detected to assess the composition of the gut flora. LR 17938 administration decreased the NEC-induced increase in intestinal scores, mortality rate, gut damage, the MDA level, and TNF-α and IL-1β expressions. Besides, LR 17938 improved the survival rate of NEC mice. Moreover, LR 17938 administration improved gut permeability levels, SOD activity and the bacterial translocation, ameliorated the expression of TJ proteins, and improved the gut microbiota compared with those of NEC mice. LR 17938 reduced intestinal inflammation and played a protective role in a neonatal animal model of NEC, possibly by regulating oxidative stress and exerting a protective effect on the gut mucosal and immune barriers. · Our research indicated a protective effect of LR 17938 on gut barrier function in NEC mice.. · LR 17938may affect the diversity of gut flora, which are known to target beneficial bacteria.. · LR 17938 protected gut barrier function in the NEC pups by improving gut permeability..

The administration of amnion-derived multipotent cell secretome ST266 protects against necrotizing enterocolitis in mice and piglets.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants and is steadily rising in frequency. Patients who develop NEC have a very high mortality, illustrating the importance of developing novel prevention or treatment approaches. We and others have shown that NEC arises in part from exaggerated signaling via the bacterial receptor, Toll-like receptor 4 (TLR4) on the intestinal epithelium, leading to widespread intestinal inflammation and intestinal ischemia. Strategies that limit the extent of TLR4 signaling, including the administration of amniotic fluid, can reduce NEC development in mouse and piglet models. We now seek to test the hypothesis that a secretome derived from amnion-derived cells can prevent or treat NEC in preclinical models of this disease via a process involving TLR4 inhibition. In support of this hypothesis, we show that the administration of this secretome, named ST266, to mice or piglets can prevent and treat experimental NEC. The protective effects of ST266 occurred in the presence of marked TLR4 inhibition in the intestinal epithelium of cultured epithelial cells, intestinal organoids, and human intestinal samples ex vivo, independent of epidermal growth factor. Strikingly, RNA-seq analysis of the intestinal epithelium in mice reveals that the ST266 upregulates critical genes associated with gut remodeling, intestinal immunity, gut differentiation. and energy metabolism. These findings show that the amnion-derived secretome ST266 can prevent and treat NEC, suggesting the possibility of novel therapeutic approaches for patients with this devastating disease.NEW & NOTEWORTHY This work provides hope for children who develop NEC, a devastating disease of premature infants that is often fatal, by revealing that the secreted product of amniotic progenitor cells (called ST266) can prevent or treat NEC in mice, piglet, and "NEC-in-a-dish" models of this disease. Mechanistically, ST266 prevented bacterial signaling, and a detailed transcriptomic analysis revealed effects on gut differentiation, immunity, and metabolism. Thus, an amniotic secretome may offer novel approaches for NEC.

Novel pharmacological inhibition of JMJD3 improves necrotizing enterocolitis by attenuating the inflammatory response and ameliorating intestinal injury

Necrotizing enterocolitis (NEC), an acute intestinal inflammatory disease of premature infants, is one of the leading causes of death in neonates. Effective measures for clinical treatment are limited and there is a pressing need in searching for new therapeutic strategies. Jumonji domain-containing protein D3 (JMJD3), a histone H3 lysine 27 (H3K27) demethylase plays a proinflammatory role in sepsis and neuroinflammation. However, whether JMJD3 is involved in the pathogenesis of NEC has not been elucidated. Here we report that overexpressed JMJD3 was revealed in the intestine of NEC patients by bioinformatic analysis. Moreover, upregulated JMJD3 and suppressed H3K27me3 were detected in both NEC patients and neonatal mice subjected to experimental NEC. Importantly, administration of GSK-J4, a specific JMJD3 inhibitor, rescued neonatal mice from NEC-associated lethality by suppressing proinflammatory response with attenuated IL-6, TNF-α, and MCP-1 levels and ameliorating intestinal injury with reversed claudin-1, occludin, and E-cadherin expression. Remarkably, administration of GSK-J4 attenuated intestinal injury by inhibiting activation of intestinal necroptosis in NEC mice. Administration of GSK-J4 regulated intestinal inflammation via NF-κB and JAK2/STAT3 pathway. These results indicate that JMJD3 is involved in the development of NEC and inhibition of JMJD3 overexpression by mean of GSK-J4 could be a potential therapeutic approach in the prevention and treatment of NEC.

Sulforaphane Ameliorates the Intestinal Injury in Necrotizing Enterocolitis by Regulating the PI3K/Akt/GSK-3β Signaling Pathway.

Objective Necrotizing enterocolitis (NEC) is a serious neonatal disease; this study aims to investigate the role of sulforaphane (SFN) in NEC-induced intestinal injury. Methods An animal model of NEC was established in newborn mice and intragastrically administrated with SFN; then, the general status and survival of the mice were observed. H&E staining was used to observe the pathological changes of intestinal tissues. ELISA, immunohistochemical staining, and flow cytometry assays were used to detect the levels of inflammatory factors, including TNF-α, IL-6, and IL-17, the expression of Bax, Bcl-2, TLR4, and NF-κB, and the percentages of the Th17 and Treg cells, respectively. GSK-3β expression levels were measured by immunofluorescence. IEC-6 and FHC cells were induced with LPS to mimic NEC in vitro and coincubated with SFN; then, the inflammatory factor levels and cell apoptosis rate were detected. Finally, Western blot was used to assess the expression of PI3K/Akt/GSK-3β pathway-related proteins in vitro and in vivo. Results SFN improved the survival rate of NEC mice during modeling, alleviated the severity of the intestinal injury, and reduced the proportion of Th17/Treg cells. SFN could inhibit TLR4 and NF-κB levels, decrease the release of inflammatory factors TNF-α and IL-6, suppress Bax expression, increase Bcl-2 expression, and inhibit apoptosis both in in vitro and in vivo models of NEC. Meanwhile, SFN regulated the expression of PI3K/Akt/GSK-3β pathway-related proteins in vitro and in vivo. Conclusion SFN relieved the inflammatory response and apoptosis by regulating the PI3K/Akt/GSK-3β signaling pathway, thereby alleviating NEC in model mice and cells.

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.