Necrotizing Enterocolitis Rat Research Articles

Silencing miR-155–5p expression improves intestinal damage through inhibiting inflammation and ferroptosis in necrotizing enterocolitis

BackgroundNecrotizing enterocolitis (NEC) is a condition characterized by acquired damage to the mucosal lining, predominantly affecting premature infants. Bioinformatics assessments uncovered a notable rise in miR-155–5p expression in the intestinal tissues of infants suffering from NEC. Nevertheless, the development of NEC's underlying mechanisms and the role of miR-155–5p are still not well understood. This research aimed to explore the role of miR-155–5p in NEC and to elucidate its underlying mechanisms. MethodsTo replicate NEC in vitro, lipopolysaccharide (LPS) was employed, whereas an in vivo rat model of NEC was established using formula feeding and exposure to hypoxia. Subsequently, levels of inflammatory cytokines, cell survival, and apoptosis rates were assessed. Various biochemical indicators such as glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were measured utilizing a purchased diagnostic kit. For the assessment of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) within FHC cells, analysis by flow cytometry was conducted. Additionally, the technique of Western blotting was utilized to analyze the levels of ferroptosis-associated proteins. Moreover, hematoxylin and eosin (H&E) staining was carried out to observe the histopathological alterations in the intestinal tissue samples from rats with necrotizing enterocolitis (NEC). ResultsReducing miR-155–5p improved the survival of FHC cells exposed to LPS, decreased cell apoptosis, inflammation, and ferroptosis, and mitigated intestinal damage in NEC rats. Furthermore, SLC7A11 was found to be a direct target of miR-155–5p. The inhibition of miR-155–5p decreased LPS-induced inflammation and ferroptosis in both FHC cells and NEC rats by promoting SLC7A11 expression. This effect was evidenced by increased levels of ferroptosis-related proteins FTH1 and GPX4, decreased COX-2 and ACSL4 levels, lower lipid peroxidation marker MDA, reduced antioxidant markers GSH, SOD, and CAT, fewer IL-6 and TNF-α, and suppression of the IκBα/NF-κB p65 signaling pathway. ConclusionsIn conclusion, reducing miR-155–5p could improve intestinal damage in NEC by inhibiting inflammation and ferroptosis. These findings may provide theoretical insights for the development of new therapies for NEC.

Bacteroides fragilis alleviates necrotizing enterocolitis through restoring bile acid metabolism balance using bile salt hydrolase and inhibiting FXR-NLRP3 signaling pathway

ABSTRACT Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants with no specific treatments available. We aimed to identify the molecular mechanisms underlying NEC and investigate the therapeutic effects of Bacteroides fragilis on NEC. Clinical samples of infant feces, bile acid-targeted metabolomics, pathological staining, bioinformatics analysis, NEC rat model, and co-immunoprecipitation were used to explore the pathogenesis of NEC. Taxonomic characterization of the bile salt hydrolase (bsh) gene, enzyme activity assays, 16S rRNA sequencing, and organoids were used to explore the therapeutic effects of B. fragilis on NEC-related intestinal damage. Clinical samples, NEC rat models, and in vitro experiments revealed that total bile acid increased in the blood but decreased in feces. Moreover, the levels of FXR and other bile acid metabolism-related genes were abnormal, resulting in disordered bile acid metabolism in NEC. Taurochenodeoxycholic acid accelerated NEC pathogenesis and taurodeoxycholate alleviated NEC. B. fragilis displayed bsh genes and enzyme activity and alleviated intestinal damage by restoring gut microbiota dysbiosis and bile acid metabolism abnormalities by inhibiting the FXR-NLRP3 signaling pathway. Our results provide valuable insights into the therapeutic role of B. fragilis in NEC. Administering B. fragilis may substantially alleviate intestinal damage in NEC.

Photoacoustic imaging for non-invasive assessment of biomarkers of intestinal injury in experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is an often-lethal disease of the premature infant intestinal tract, exacerbated by significant diagnostic difficulties. In NEC, the intestine exhibits hypoperfusion and dysmotility, contributing to disease pathogenesis. However, these features cannot be accurately and quantitively assessed with current imaging modalities. We have previously demonstrated the ability of photoacoustic imaging (PAI) to non-invasively assess intestinal tissue oxygenation and motility in a healthy neonatal rat model. In this first-in-disease application, we evaluated NEC using PAI to assess intestinal health biomarkers in an experimental model of NEC. NEC was induced in neonatal rats from birth to 4-days. Healthy breastfed (BF) and NEC rat pups were imaged at 2- and 4-days. Intestinal tissue oxygen saturation was measured with PAI, and NEC pups showed significant decreases at 2- and 4-days. Ultrasound and PAI cine recordings were used to capture intestinal peristalsis and contrast agent transit within the intestine. Intestinal motility, assessed using computational intestinal deformation analysis, demonstrated significant reductions in both early and established NEC. NEC damage was confirmed with histology and dysmotility was confirmed by small intestinal transit assay. This preclinical study presents PAI as an emerging diagnostic imaging modality for intestinal disease assessment in premature infants. Necrotizing enterocolitis (NEC) is a devastating intestinal disease affecting premature infants with significant mortality. NEC presents significant clinical diagnostic difficulties, with limited diagnostic confidence complicating timely and effective interventional efforts. This study is an important foundational first-in-disease preclinical study that establishes the utility for PAI to detect changes in intestinal tissue oxygenation and intestinal motility with NEC disease induction and progression. This study demonstrates the feasibility and exceptional promise for the use of PAI to non-invasively assess oxygenation and motility in the healthy and diseased infant intestine.

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.

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.

Astragaloside IV regulates TL1A and NF-κB signal pathway to affect inflammation in necrotizing enterocolitis

AimThis study aims to explore the possible effect of Astragaloside IV (AS-IV) on necrotizing enterocolitis (NEC) neonatal rat models and verify the possible implication of TNF-like ligand 1 A (TL1A) and NF-κB signal pathway. MethodsNEC neonatal rat models were established through formula feeding, cold/asphyxia stress and Lipopolysaccharide (LPS) gavage method. The appearance, activity and skin as well as the pathological status of rats subjected to NEC modeling were assessed. The intestinal tissues were observed after H&E staining. The expression of oxidative stress biomarkers (SOD, MDA and GSH-Px) and inflammatory cytokines (TNF-α, IL-1β and IL-6) were detected by ELISA and qRT-PCR. Western blotting and immunohistochemistry were applied to detect expressions of TL1A and NF-κB signal pathway-related proteins. Cell apoptosis was assessed by TUNEL. ResultsNEC neonatal rat models were established successfully, in which TL1A was highly expressed and NF-κB signal pathway was activated, while TL1A and NF-κB signal pathway can be suppressed by AS-IV treatment in NEC rats. Meanwhile, inflammatory response in intestinal tissues was increased in NEC rat models and AS-IV can attenuate inflammatory response in NEC rats through inhibiting TL1A and NF-κb signal pathway. ConclusionAS-IV can inhibit TL1A expression and NF-κb signal pathway to attenuate the inflammatory response in NEC neonatal rat models.

Astaxanthin Alleviates Inflammatory Response in Neonatal Necrotizing Enterocolitis Rats by Regulating NOD2/TLR4 Pathway.

Necrotizing enterocolitis (NEC) is often associated with exaggerated activation of inflammatory response. Astaxanthin has been shown in studies to have a positive and advantageous effect on anti-inflammatory response. Hence, it is of great significance to study the protective effect of astaxanthin in NEC disease and its molecular mechanism. The present study was to investigate whether astaxanthin attenuates NEC rats and to explore its potential mechanism. Material and Methods. Hematoxylin-eosin staining was used to observe the pathological change of the intestinal tissue in NEC rats. Subsequently, we determined the anti-oxidative stress, anti-apoptosis, and anti-inflammation in astaxanthin with enzyme-linked immunosorbent assay kits, TUNEL staining, western blot, and immunohistochemistry assay. Furthermore, we added nucleotide-binding oligomerization domain 2 (NOD2) inhibitor to certify the molecular pathway of the astaxanthin in NEC rats. Astaxanthin improved the pathological changes of the intestinal tissues. It restrained inflammation, oxidative stress, and protected cells from apoptosis in the intestinal tissue and serum of the NEC rats. Moreover, astaxanthin enhanced NOD2, whereas it suppressed toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB) pathway-related proteins. Apart from that, the NOD2 inhibitor offset the protective effect of the astaxanthin towards the NEC rats. The present study indicated that astaxanthin alleviated oxidative stress, inflammatory response, and apoptosis in NEC rats by enhancing NOD2 and inhibiting TLR4 pathway.

Suppressive role of vascular endothelial growth factor on intestinal apoptosis in induced necrotizing enterocolitis in rats.

Necrotizing enterocolitis (NEC) is a devastating disease that can cause mortality in preterm babies. NEC may develop through an apoptotic pathway that is known to be inhibited by vascular endothelial growth factor (VEGF). This study determined whether VEGF exerted a protective effect against the development of NEC and apoptosis in rats. To determine the effect of VEGF in NEC rats, neonatal rats were randomized into 4 groups: the control group, the NEC group, the NEC + intraperitoneal VEGF (50 ng/kg) group (NEC + VEGF IP group), and the NEC + oral VEGF (50 ng/kg) group (NEC + VEGF OR group). NEC was induced by lipopolysaccharide/hypoxia and cold stress. The animals were sacrificed 72 hours later. After laparotomy, we obtained a region of the proximal small bowel from the ileocecal valve about 18 cm in length. The NEC histological grade, apoptosis histological score, and caspase-3 activity were lower in the NEC + VEGF IP and OR groups than in the NEC group. In the NEC + VEGF IP and OR groups, the messenger RNA expression of apoptotic and inflammatory genes, such as Bax, NF-κB, p53, Fas, FasL, and PAF-R, but not that of Bcl-2, was decreased, as was the Bax/Bcl-2 protein ratio. Histological analysis revealed that the apoptosis-blocking effect of VEGF was more effective in the NEC + VEGF IP group than in the NEC + VEGF OR group. We identified apoptotic and inflammatory genes to confirm the preventive effect of VEGF pretreatment on NEC in rats. This study presents a novel approach to prevent apoptosis via VEGF pretreatment in rats with lipopolysaccharide/hypoxia-induced NEC.

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.

Resveratrol ameliorates necrotizing enterocolitis in neonatal rats through regulation of inflammatory and apoptotic pathways

Purpose: To determine the efficacy of resveratrol in mitigating necrotizing enterocolitis (NEC) in neonatal rats.Methods: Necrotizing enterocolitis (NEC) was induced in neonatal rats using hypoxia and hypothermia. At the completion of treatment, the intestinal tissues of the rats were isolated for evaluation of various biochemical parameters.Results: There was significant increase in the levels of proinflammatory cytokines (TNF-α, IL-6 and IL-1β) and oxidative stress markers (MDA, xanthine oxidase and nitric oxide) in intestinal tissues of NEC rats (p &lt; 0.05). However, resveratrol treatment led to significant decrease in the levels of cytokines and oxidative stress parameters, relative to the NEC group (p &lt; 0.05). Furthermore, Western blotting resultsshowed up-regulation in protein expressions of inflammatory cytokines in NEC rats. However, the protein expressions of inflammatory cytokines were down-regulated in the NEC rats on treatment with resveratrol. Moreover, resveratrol reversed the NEC-induced up-regulations of Bax and caspase-3, as well as NEC-mediated down-regulation of Bcl-2.Conclusion: These results demonstrate that resveratrol mitigates NEC-induced intestinal damage in neonatal rats via anti-inflammatory, anti-apoptotic and antioxidant mechanisms of action. Therefore, resveratrol is a potential therapeutic agent for NEC.

The protective effects of Anakinra in a neonatal rat model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a commonly seen life-threatening condition in newborns characterized by ischemic necrosis. This study aimed to investigate anakinra's effects, an interleukin-1 receptor antagonist, on oxidative stress, inflammation, and tissue necrosis in an NEC rat model. Forty Wistar albino pups were divided into four groups randomly as follows; group 1, control group; group 2, anakinra-treated control group; group 3, NEC group; and group 4, NEC and anakinra treatment group. The rats were given hyperosmolar formula feeding, and they were exposed to hypoxia after cold stress at +4 °C and oxygen in order to create the NEC model. On the 4th day of the experiment, the pups were decapitated, and the intestinal tissues were resected for biochemical and histopathologic examination. Microscopic injury scores and apoptotic indexes were higher in group 3 than the control group (p < 0.001, p = 0.002, respectively), and there was a significant decrease after anakinra. Interleukin 1β and caspase-3 levels increased with NEC and decreased significantly after administration of anakinra (p = 0.006, p = 0.004, respectively). Malondialdehyde and glutathione peroxidase levels also increased compared with the control group (p = 0.019, p = 0.002, respectively). In this experimental study, we found that anakinra had antiinflammatory and antioxidant effects and was protective against intestinal injury and apoptosis.

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.

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..

Causative role of mast cell and mast cell-regulatory function of disialyllacto-N-tetraose in necrotizing enterocolitis

Necrotizing enterocolitis (NEC) remains a fatal gastrointestinal disorder in neonates. Disialyllacto-N-tetraose (DSLNT), a function-unclear human milk-derived hexasaccharide, shows anti-NEC potential in previous animal studies. This study is aimed to explore the role of mast cell (MC), a fundamental cell type of mucosal immune system and protective DSLNT in regulating pathological process of NEC. For this purpose, infantile intestinal-tissues were collected from NEC neonates for examination of MCs and its proteases-positive cells. MC accumulation and MC-specific proteases (chymase, tryptase and dipeptidyl peptidase I) were firstly found in lesioned area of NEC infants in-vivo. Subsequent in-situ experiments on neonatal ileum segments showed that purified MC-chymase induced a destructive epithelial layer shedding from basement and microvascular endothelium damage in infantile intestinal segments. Human foreskin MC-activation model was established and DSLNT were applied; MC products (histamine and MC-proteases) were used as MC activation/degranulation indicators. In this in-vitro model, DSLNT pretreatment suppressed release of histamine, chymase and tryptase by MC to the tissue supernatants during lipopolysaccharide or complement C5a stimulation. Newborn rats were formula-hand-fed with or without DSLNT supplement and exposed to hypoxia/cold-stress to induce experimental-NEC-model. In NEC rats, DSLNT supplementation reduced the incidence and pathological scores of NEC, inhibited local accumulation of MC and reduced cytokines (IL-1β, IL-6 and TNF-α) levels in the ileum of rats. In conclusion, MC was causally implicated in epithelium barrier failure in pathogenesis of NEC. DSLNT favorably modulated MC homeostasis by regulating MC degranulation/accumulation, contributing to attenuated NEC. This indicated novel pathomechanisms and potential targets of NEC.

P0639 / #1836: PROBIOTICS FOR TREATMENT OF NECROTIZING ENTEROCOLITIS IN A MURINE MODEL

Aims & Objectives: Necrotizing enterocolitis (NEC) is one of the devastating gastrointestinal disorders. Probiotics are believed to decrease the risk of NEC and it has also been used for its treatment Methods: A total of 142 Sprague-Dawley (SD) rats were used to secure a total of 80 neonatal pubs. NEC was induced through exposing neonatal rats obtained via caesarean section after 18-20 days gestation (prematurity stage) to asphyxia and cold stress. Pubs were randomly divided into the following four groups: Group I (n=20) were hypoxia formula feeding (FFH) rat model of induced NEC, who have no therapy. Group II (n=20) included 20 rats of FFH-rat model of induced NEC, who underwent probiotic treatment. Group III (n=20) were rat models FFH-rat model of induced NEC, treated by oral antibiotics. Group IV (n=20) included premature, breast-fed rats, serving as controls. Seven days after birth, all rats were sacrificed, intestinal injury and TNF-α-, IL-18-, as well as iNOS2-expression in the intestine and liver were assessed. Results: Probiotics reduced the histological score and thus significantly reduced the rate of NEC-like intestinal injury. NEC was associated with increased Hepatic TNF-α and IL-18 positive cells. Moreover, the intestinal flushes level of TNF-α in NEC rats were increased in correlation to the controls. This high level of intestinal TNF-α and the other inflammatory liver parameters were significantly decreased in the probiotics treated model. Conclusions: This experimental study demonstrated that probiotics could treat NEC in preterm neonatal rats. The mechanism for this action might be associated with the suppressed expression of pro inflammatory mediators.

Sialylated human milk oligosaccharides prevent intestinal inflammation by inhibiting toll like receptor 4/NLRP3 inflammasome pathway in necrotizing enterocolitis rats

BackgroundNecrotizing enterocolitis (NEC) remains a fatal gastrointestinal disorder in neonates and has very limited therapeutic options. Sialylated human milk oligosaccharides (SHMOs) improve pathological changes in experimental NEC models. The objectives of this study were to investigate the involvement of NLRP3 inflammasome in NEC pathology and to explore the effects of SHMOs on toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB)/NLRP3 inflammatory pathway in experimental NEC.MethodsThe intestinal-tissue segments were collected from NEC infants, NLRP3 and caspase-1 positive cell were examined by immunohistochemistry. Newborn rats were hand-fed with formula containing or non-containing SHMOs (1500 mg/L) and exposed to hypoxia/cold stress to induce experimental NEC. The NEC pathological scores were evaluated; ileum protein expression of membrane TLR4 (mTLR4), inhibitor κB-α (IκB-α), NF-κB p65 subunit and phospho-NF-κB p65, as well as NLRP3 and caspase-1 were analyzed; ileum concentrations of interleukin-1β, interleukin-6, tumor necrosis factor-α (TNF-α) were also measured. Human colon epithelial Caco-2 cells were pre-treated with or without SHMOs and stimulated with TLR4 activator, lipopolysaccharide. Cell viabilities, mitochondrial membrane potential and supernatant matrix metalloprotease 2 (MMP-2) activities were analyzed.ResultsIncreased frequencies of NLRP3 and caspase-1 positive cells were found in the lamina propria of damaged intestinal area of NEC neonates. SHMOs supplementation reduced NEC incidence and pathological damage scores of rats challenged with hypoxia/cold stress. Accumulation of interleukin-1β, interleukin-6 and TNF-α in NEC group were attenuated in SHMOs + NEC group. Protein expression of mTLR4, NLRP3 and caspase-1 were elevated, cytoplasmic IκB-α were reduced, nuclear phospho-NF-κB p65 were increased in the ileum of NEC rats. SHMOs supplementation ameliorated the elevation of mTLR4, NLRP3 and caspase-1, restored IκB-α in the cytoplasmic fraction and reduced phospho-NF-κB p65 in the nuclear fraction in the ileum of NEC rats. SHMOs pre-treatment improved Caco-2 cell viability, mitigated loss of mitochondrial membrane potential and modulated MMP-2 activities in the presence of lipopolysaccharide in-vitro.ConclusionsThis study provided clinical evidence of involvement of NLRP3 inflammasome in NEC pathology, and demonstrated the protective actions of SHMOs might be owing to the suppression of TLR4/NF-κB/NLRP3-mediated inflammation in NEC.

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.

Let-7d-5p suppresses inflammatory response in neonatal rats with necrotizing enterocolitis via LGALS3-mediated TLR4/NF-κB signaling pathway.

Necrotizing enterocolitis (NEC) is an acute intestinal condition accounting for severe mortality and morbidity in preterm infants. This study aimed to identify the possible roles of let-7d-5p in neonatal rats with NEC. The differentially expressed genes (DEGs) related to NEC were initially screened in silico. After establishment of NEC rat models, measurement of the expression of let-7d-5p, galectin-3 (LGALS3), Toll-like receptor 4 (TLR4), and nuclear factor-κB (NF-κB) as well as proinflammatory cytokines (TNF-α, IL-1β, and IL-6) was conducted. The interaction between let-7d-5p and LGALS3 or argonaute-2 (AGO2) was identified. Gain- and loss-of-function approaches were then performed in an attempt to investigate the regulatory roles of let-7d-5p and LGALS3 in inflammation and cell apoptosis in NEC neonatal rats. Let-7d-5p was poorly expressed, whereas LGALS3, TLR4, and NF-κB were highly expressed, in the intestinal tissues of NEC rats. Overexpression of let-7d-5p resulted in decreased levels of proinflammatory factors in the intestinal tissues of NEC rats. Through sequential experimentation, let-7d-5p was identified to target LGALS3 and bind to AGO2. In addition, LGALS3 silencing or LPS treatment blocked the TLR4/NF-κB signaling pathway, thereby suppressing intestinal epithelial cell apoptosis and inflammation in NEC. Collectively, let-7d-5p might exercise its inhibitory properties in the inflammatory response and intestinal epithelial cell apoptosis in NEC neonatal rats via inactivation of the LGALS3-dependent TLR4/NF-κB signaling pathway.

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.