MiRNA-192-5p-targeted activated leukocyte cell adhesion molecule improved inflammatory injury of neonatal necrotizing enterocolitis.

BACKGROUND: Neonatal necrotizing enterocolitis (NEC) is a common gastrointestinal emergency in neonates. MiRNA-192-5p was found associated with ulcerative colitis (UC) progression, also with aberrant expression in intestinal cancer tissue. However, the effects of miRNA-192-5p on NEC have not been reported. METHODS: Based on the bioinformatics analysis of the GEO dataset, miR-192-5p was identified as the differentially expressed miRNA in NEC, and activated leukocyte cell adhesion molecule (ALCAM) was predicted as its target. After that, in vitro, rat intestinal epithelial cell-6 (IEC-6) were stimulated with LPS to construct a cell model of NEC. IEC-6 cells were transfected with miRNA-192-5p mimics, miRNA-192-5p inhibitors, or miRNA-192-5p inhibitors + sh-ALCAM, and relevant negative control. In vivo, SD rats were treated with artificial feeding, hypoxic reoxygenation, cold stimulation, and LPS gavage to induce NEC, followed by injection of agomiR-NC or agomiRNA-192-5p. Then effects of miRNA-192-5p on NEC model IEC-6 cell viability, apoptosis, ALCAM expression, Interleukin (IL)-1ß and IL-6 levels, intestinal injury, intestinal permeability were detected. RESULTS: MiRNA-192-5p expression was downregulated in NEC IEC-6 cells, whose overexpression increased IEC-6 cell viability. MiRNA-192-5p inhibitors increased IL-1ß, IL-6 levels and promoted IEC-6 cell apoptosis. MiRNA-192-5p targeting of ALCAM decreased ALCAM expression, IL-1ß, and IL-6 levels. AgomiRNA-192-5p decreased ALCAM, IL-1ß, and IL-6 levels in intestinal tissue and pathological damage and increased miRNA-192-5p levels. CONCLUSION: MiR-192-5p protects against intestinal injury by inhibiting ALCAM-mediated inflammation and intestinal epithelial cells, which would provide a new idea for NEC treatment.

Necrotizing enterocolitis: current understanding of the prevention and management.

Necrotizing enterocolitis (NEC) is one of the diseases in neonates, with a high morbidity and mortality rate, especially in preterm infants. This review aimed to briefly introduce the latest epidemiology, susceptibility factors, and clinical diagnosis and presentation of NEC. We also organized new prevention strategies by risk factors according to different pathogeneses and then discussed new treatment methods based on Bell's staging and complications, and the classification of mild to high severity based on clinical and imaging manifestations. Such a generalization will help clinicians and researchers to gain a deeper understanding of the disease and to conduct more targeted classification, grading prevention, and exploration. We focused on prevention and treatment of the early and suspected stages of NEC, including the discovery of novel biomarkers and drugs to control disease progression. At the same time, we discussed its clinical application, future development, and shortcomings.

Patent ductus arterious and increased conjugated bilirubin in the second week after birth are independent risk factors for necrotizing enterocolitis in preterm infants: an observational study.

BACKGROUND: Neonatal necrotizing enterocolitis (NEC) is a common critical illness of the gastrointestinal system in neonatal intensive care units with complex causes. We want to explore effects of serum-conjugated bilirubin on the occurrence of NEC in preterm infants. METHODS: A retrospective study of clinical case data of premature infants from 2017 to 2020 in the Department of pediatrics of the First Affiliated Hospital of Nanjing Medical University was conducted. Among these, 41 were diagnosed with NEC. After screening, 2 cases were excluded because of incomplete data. Propensity-matching score (PSM) was performed according to the ratio of 1:2(2 preterm infants in the NEC group were not matched), and finally, 37 cases were in the NEC group (average time to diagnosis was 18.9 days), and 74 cases in the non-NEC group. We compared the difference between the NEC and non-NEC groups in early serum-conjugated bilirubin and total bilirubin levels (time points: the first day of birth, 1 week after birth, 2 weeks after birth). RESULTS: (1) The changing trend of conjugated bilirubin was different between the two groups(F = 4.085, P = 0.019). The NEC group's serum-conjugated bilirubin levels gradually increased ([Formula: see text] ± s:12.64±2.68; 17.11±4.48; 19.25±11.63), while the non-NEC group did not show a continuous upward trend ([Formula: see text] ± s:13.39±2.87; 15.63±3.75; 15.47±4.12). (2) Multiple analyses showed that patent ductus arteriosus(PDA) (odds ratio[OR] = 5.958, 95%confidence interval[CI] = 2.102 ~ 16.882) and increased conjugated bilirubin in the 2nd week (OR = 1.105, 95%CI = 1.013 ~ 1.206) after birth were independent risk factors for NEC. CONCLUSIONS: The body had already experienced an elevation of conjugated bilirubin before the occurrence of NEC. The change of early conjugated bilirubin may be an important factor in the occurrence of NEC.

Bioinformatics analysis of potential key genes and pathways in neonatal necrotizing enterocolitis.

OBJECTIVE: To detect differentially expressed genes in patients with neonatal necrotizing enterocolitis (NEC) by bioinformatics methods and to provide new ideas and research directions for the prevention, early diagnosis and treatment of NEC. METHODS: Gene chip data were downloaded from the Gene Expression Omnibus database. The genes that were differentially expressed in NEC compared with normal intestinal tissues were screened with GEO2R. The functions, pathway enrichment and protein interactions of these genes were analyzed with DAVID and STRING. Then, the core network genes and significant protein interaction modules were detected using Cytoscape software. RESULTS: Overall, a total of 236 differentially expressed genes were detected, including 225 upregulated genes and 11 downregulated genes, and GO and KEGG enrichment analyses were performed. The results indicated that the upregulated differentially expressed genes were related to the dimerization activity of proteins, while the downregulated differentially expressed genes were related to the activity of cholesterol transporters. KEGG enrichment analysis revealed that the differentially expressed genes were significantly concentrated in metabolism, fat digestion and absorption pathways. Through STRING analysis, 9 key genes in the protein network interaction map were identified: EPCAM, CDH1, CFTR, IL-6, APOB, APOC3, APOA4, SLC2A and NR1H4. CONCLUSION: Metabolic pathways and biological processes may play important roles in the development of NEC. The screening of possible core targets by bioinformatics is helpful in clarifying the pathogenesis of NEC at the gene level and in providing references for further research.

Blockage of NLRP3 inflammasome activation ameliorates acute inflammatory injury and long-term cognitive impairment induced by necrotizing enterocolitis in mice.

BACKGROUND: Necrotizing enterocolitis (NEC) is an inflammatory gastrointestinal disease in premature neonates with high mortality and morbidity, while the underlining mechanism of intestinal injury and profound neurological dysfunction remains unclear. Here, we aimed to investigate the involvement of NLPR3 inflammasome activation in NEC-related enterocolitis and neuroinflammation, especially long-term cognitive impairment, meanwhile, explore the protective effect of NLRP3 inhibitor MCC950 on NEC in mice. METHODS: NLRP3 inflammasome activation in the intestine and brain was assessed in the NEC mouse model, and NLRP3 inhibitor MCC950 was administrated during the development of NEC. Survival rate, histopathological injury of the intestine and brain, and expression of mature IL-1ß and other pro-inflammatory cytokines were analyzed. Long-term cognitive impairment was evaluated by behavioral test. RESULTS: The expression of NLRP3 and mature IL-1ß in the intestine and brain was greatly upregulated in NEC mice compared to the controls. MCC950 treatment efficiently improved NEC survival rate, reduced intestinal and brain inflammation, and ameliorated the severity of pathological damage in both organs. Additionally, in vivo blockage of NLRP3 inflammasome with MCC950 in early life of NEC pups potently protected against NEC-associated long-term cognitive impairment. CONCLUSIONS: Our findings suggest that NLRP3 inflammasome activation participates in NEC-induced intestinal and brain injury, and early intervention with NLRP3 inhibitor may provide beneficial therapeutic effect on NEC infants.

Activated M1 macrophages suppress c-kit expression via TNF-α-mediated upregulation of miR-222 in Neonatal Necrotizing Enterocolitis.

BACKGROUND: Activation of intestinal macrophages is implicated in the pathogenesis of neonatal necrotizing enterocolitis (NEC), yet its precise mechanisms remain unclear. OBJECTIVE: The purpose of this study is to investigate the role of macrophages and TNF-α via an inflammatory MicroRNA in NEC. MATERIALS AND METHODS: Immunofluorescence (IF) staining of CD68, iNOS, and Arg-1 was employed to identify phenotypes of macrophage in the intestines of NEC infants and NEC mice. Expression of TNF-α, c-kit, and miR-222 was evaluated by qRT-PCR, Western blot, and immunochemical staining from the tissue samples. RESULTS: Large number of M1 macrophage infiltration was found in the NEC intestines. Expression of CD68, iNOS, and TNF-α were significantly increased, while c-kit was decreased distinctly in the NEC group. In the early phase of NEC mouse model, inhibition of M1 macrophages reduced the incidence of NEC and intestinal inflammation. We found that TNF-α upregulated the expression of miRNA-222 and inhibited the expression of c-kit. Conversely, such decrease of c-kit expression could be reversed by miR-222 antagonists. Furtherly, dual-luciferase assay confirmed that c-kit can be inhibited by miR-222 directly. CONCLUSION: Macrophages activation in NEC intestine results in an increased inflammatory response and TNF-α production, accompanied with miR-222 upregulation and c-kit suppression. Modulations of M1 macrophages, TNF-α or miR-222 may be potential therapeutic targets for NEC treatment.

The Role of Gastrointestinal-Related Fatty Acid-Binding Proteins as Biomarkers in Gastrointestinal Diseases.

The fatty acid-binding proteins play a major role in intracellular transportation of long-chain fatty acids. Nine fatty acid-binding proteins have been identified, with each having individual tissue-specific functions in addition to regulation of fatty acids. This review focuses on the three fatty acid-binding proteins found in the gastrointestinal tract and discusses their role as diagnostic or disease monitoring markers in neonatal necrotizing enterocolitis, acute mesenteric ischemia, celiac disease, and inflammatory bowel disease. Of these three fatty acid-binding proteins, intestinal fatty acid-binding protein is of the most interest due to its exclusive expression in the gastrointestinal tract. The elevation of intestinal fatty acid-binding protein in blood and urine reflects enterocyte damage, regardless of the underlying cause. The short half-life of intestinal fatty acid-binding protein also means it is a relatively sensitive marker. In contrast, there is currently less evidence to support liver fatty acid-binding protein and ileal bile acid-binding protein as sensitive biomarkers in these conditions. More extensive studies with specific endpoints are required to validate the roles of these fatty acid-binding proteins in gastrointestinal diseases.

Changes of long non-coding RNA expression profiles in intestinal tissues from neonatal necrotizing enterocolitis. / è‚ ç»„ç»‡é•¿é“¾éžç¼–ç RNAè¡¨è¾¾è°±åœ¨æ–°ç”Ÿå„¿åæ­»æ€§å°è‚ ç»“è‚ ç‚Žç»„ç»‡ä¸­çš„å˜åŒ–.

OBJECTIVES: Long non-coding RNAs (lncRNAs) were involved in the development and regulation of necrotizing enterocolitis (NEC) in premature infants. To investigate the changes of lncRNA expression profile in intestinal tissues of NEC for its possible mechanisms. METHODS: Intestinal samples were collected from 11 patients with NEC who needed surgery(the NEC group), and 7 from neonatal non-NEC patients with surgery (the Control group).LncRNA's changes in intestinal samples (3 in the Control group and 3 in the NEC group) were analyzed with high-throughput sequencing.Part of the remaining samples were detected by real-time polymerase chain reaction (real-time PCR), and the results were used to validate the results of high-throughput sequencing. Gene Ontology (GO) analysis and KEGG signaling pathway analysis were performed on differentially expressed genes. RESULTS: There were 5 257 different lncRNAs between the control group and the NEC group. The results of up-regulated lncRNAs (NONHSAG008675.3, NONHSAG020715.2, NONHSAG038187.2) and down-regulated lncRNA (NONHSAG028744.3) were confirmed to be consistent with the results of high-throughput sequencing. Expressions of DUOX2, IL-6, TNF, and SAA1 were up-regulated in intestinal tissues of NEC. GO analysis showed that the different lncRNAs were involved in regulation of stimulation, molecular junction and function, and signal transduction and transcription. KEGG analysis identified mainly biological pathways involved in inflammatory bowel disease, PI3K-Akt, NF-κB, etc. CONCLUSIONS: LncRNAs might be involved in the pathogenesis of NEC and the inflammation-related lncRNAs may be one of the key factors.

Regulation of ROS-NF-κB axis by tuna backbone derived peptide ameliorates inflammation in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the most common life-threatening gastrointestinal disease encountered in the premature infant. It has been shown that the intercellular reactive oxygen species (ROS) generation activated by lipopolysaccharide involved in the nuclear factor kappa B (NF-κB) activation and pathogenesis of NEC. Here, we report that an antioxidant peptide from tuna backbone protein (APTBP) reduces the inflammatory cytokines transcription and release. APTBP directly scavenges the free radical through 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) assay. In addition, APTBP reduces the intracellular ROS level, exhibiting an antioxidant activity within cells. Remarkably, gavage with APTBP attenuates the phenotype of NEC in the mice model. Mechanically, the NF-κB activation, together with the expression of inflammatory cytokines are decreased significantly when intracellular ROS are eliminated by APTBP. Therefore, our findings demonstrated that an antioxidant peptide, APTBP, ameliorates inflammation in NEC through attenuating ROS-NF-κB axis.

Inhibition of miR-124 improves neonatal necrotizing enterocolitis via an MYPT1 and TLR9 signal regulation mechanism.

BACKGROUNDS: Necrotizing enterocolitis (NEC) was one of the main causes of morbidity and mortality in neonates. Our objective was to detect the mechanism of miR-124 in small bowel tissues of NEC. METHODS: Hematoxylin and eosin (H&E) staining was used to detect the repair of the damaged tissues in rat NEC model. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to evaluate the cell apoptosis level in intestinal tissue. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the messenger RNA (mRNA) expression level of miR-124, Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), myosin phosphatase target subunit 1 (MYPT1), and Toll-like receptor 9 (TLR9) in NEC tissues and IEC-6 cells. Luciferase reporter assay was used to verify whether ROCK1 is a direct target of miR-124. RESULTS: miR-124 was overexpressed in the NEC tissues, while ROCK1 and MYPT1 was downregulated in the NEC tissues. Inhibition of miR-124, suppressed the intestinal cell apoptosis and promoted the expression of ROCK1 and MYPT1. What is more, overexpression of miR-124 could inhibit the expression of ROCK1, TLR9, and MYPT1. Luciferase assay confirmed that miR-124 can regulate the transcriptional activity of ROCK1 through binding its 3'-UTR region. CONCLUSION: miR-124 was a promoter of NEC, which promotes the intestine cell apoptosis and inflammatory cell infiltration through the inhibition of TLR9 expression by targeting ROCK1.

Diagnostic value and disease evaluation significance of abdominal ultrasound inspection for neonatal necrotizing enterocolitis.

OBJECTIVE: To summarize abdominal plain X-rays and ultrasound characteristics of 144 cases of Neonatal Necrotizing Enterocolitis (NEC) and to analyze diagnostic value and disease evaluation significance of abdominal ultrasound inspection for NEC. METHODS: Clinical data of 144 NEC patients were retrospectively analyzed from February 2014 to December 2015. The patients were divided into suspected NEC group (N=74) and confirmed NEC group (N=70) according to amended Bell-NEC classification and diagnostic criteria. Meanwhile, we divided them into internal medicine treatment group (N=95) and surgery/death group (N=49) according to clinical prognosis and took records of their clinical manifestations, laboratory inspection results and abdominal plain X-rays and ultrasound characteristics. RESULTS: For confirmed NEC group, the detection rate of portal venous gas (PVG) and dilatation of intestine by abdominal ultrasound was obviously higher than by plain X-rays (P<0.05). Abdominal ultrasound inspection revealed that the incidence rate of dilatation of intestine, bowel wall thickening and ascites (acoustic transmission difference) of the surgery/death group was higher than that of the internal medicine treatment group by comparing risk ratio (RR) and 95% confidence interval (CI) of RR; the difference was statistically significant (P<0.05). The abdominal plain X-rays inspection only showed the result that dilatation of intestine and free intraperitoneal air was more often found in the surgery/death group (P<0.05). CONCLUSION: Compared with abdominal plain X-rays, abdominal ultrasound has certain clinical value and offers more advantages in some aspects; therefore, it can be considered as the reference index in prediction of clinical prognosis.

Probiotics and prebiotics in neonatal necrotizing enterocolitis: New opportunities for translational research.

Neonatal necrotizing enterocolitis (NEC) in premature infants has been recognized as a defined disease entity for at least four decades. Although survival has increased due to the advent of more sophisticated intensive care, incidence and long term health impacts due to NEC remain unchanged and no preventive therapy is currently available. Different probiotic strains of bacteria have been examined in their ability to prevent NEC with varied but encouraging results. Undigestable prebiotic sugars known to promote the growth of probiotic bacteria in the colon have been used in neonates with no clear benefit. The literature on NEC and probiotics is now cluttered with more reviews and meta-analyses than number of clinical trials. On the other hand, significant new information is available on microbiota and their impact on gut immunity. This review attempts to reiterate the risk factors of NEC and the pathogenesis of NEC with special reference to gut permeability. The reader is then introduced to gut microbiota, uniqueness and differences among probiotic strains, and how multiple resident flora talk to each other in the community setting in the human gut. After presenting a concise review of available clinical research results, the reader is challenged to question as to why no precise answer is available at present. Some modalities to examine the complex microflora and changes in the neonatal gut are then proposed including non-invasive methods and mathematical modeling. The review concludes by attracting the reader's attention to known immunomodulators of inflammation and injury. Justice to this review will be done only if the readers, clinical, and basic science investigators from multiple fields gather courage for a paradigm shift and embark on understanding the pathophysiology of the disease and attempt to discern the difference from equally preterm, equally vulnerable neonates that do not develop NEC. Learning about the developing microbiota in neonatal gut and its immunological impacts on the host in the face of many variables will provide a leap in our pursuit to select better, if not the best candidate probiotics, and put them to work against this stubborn disease that continues to take a toll on our precious neonates and the society.

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

AIMS: Neonatal 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 METHODS: Proteomic 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 FINDINGS: We 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. SIGNIFICANCE: Our 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.

Macrophage α7nAChR alleviates the inflammation of neonatal necrotizing enterocolitis through mTOR/NLRP3/IL-1ß pathway.

BACKGROUND: Neonatal necrotizing enterocolitis (NEC) is one of the most prevalent and severe intestinal emergencies in newborns. The inflammatory activation of macrophages is associated with the intestinal injury of NEC. The neuroimmune regulation mediated by α7 nicotinic acetylcholine receptor (α7nAChR) plays an important role in regulating macrophage activation and inflammation progression, but in NEC remains unclear. This study aims to explore the effect of macrophage α7nAChR on NEC. METHODS: Mice NEC model were conducted with high-osmolarity formula feeding, hypoxia, and cold stimulation. The α7nAChR agonist PNU-282987 and mTOR inhibitor rapamycin were treated by intraperitoneal injections in mice. The expression and distribution of macrophages, α7nAChR, and phospho-mammalian target of rapamycin (p-mTOR) in the intestines of NEC patients and mice was assessed using immunohistochemistry, immunofluorescence, and flow cytometry. The expression of NLRP3, activated caspase-1 and IL-1ß in mice intestines was detected by flow cytometry, western blot or ELISA. In vitro, the mouse RAW264.7 macrophage cell line was also cultured followed by various treatments. Expression of p-mTOR, NLRP3, activated caspase-1, and IL-1ß in macrophages was determined. RESULTS: Macrophages accumulated in the intestines and the expression of α7nAChR in the mucosal and submucosal layers of the intestines was increased in both the NEC patients and mice. The p-mTOR and CD68 were increased and co-localized in intestines of NEC patients. In vitro, α7nAChR agonist PNU-282987 significantly reduced the increase of NLRP3, activated caspase-1, and IL-1ß in macrophages. PNU-282987 also significantly reduced the increase of p-mTOR. The effect was blocked by AMPK inhibitor compound C. The expression of NLRP3, activated caspase-1, and IL-1ß was inhibited after mTOR inhibitor rapamycin treatment. In NEC model mice, PNU-282987 reduced the expression of p-mTOR, NLRP3, activated caspase-1, and IL-1ß in the intestine. Meanwhile, rapamycin significantly attenuated NLRP3 activation and the release of IL-1ß. Moreover, the proportion of intestinal macrophages and intestinal injury decreased after PNU-282987 treatment. CONCLUSION: Macrophage α7nAChR activation mitigates NLRP3 inflammasome activation by modulating mTOR phosphorylation, and subsequently alleviates intestinal inflammation and injury in NEC.

I-FABP protein/mRNA and IL-6 as biomarkers of intestinal barrier dysfunction in neonates with necrotizing enterocolitis and SPF BALB/c mouse models.

OBJECTIVE: Neonatal necrotizing enterocolitis (NEC) is a serious intestinal inflammatory disease. We investigated intestinal fatty acid binding protein (I-FABP), I-FABP mRNA, and interleukin-6 (IL-6) as potential diagnostic biomarkers in NEC. METHODS: Forty mice were subjected to hypoxic-ischemic intestinal injury, and then serum I-FABP protein and mRNA levels were quantified. Ileal tissue pathological scores were determined by hematoxylin and eosin staining. I-FABP expression levels and translocation in these tissues were detected using western blotting and immunofluorescence, respectively. Samples from 30 human neonates with NEC and 30 healthy neonates had serum I-FABP protein/mRNA and IL-6 levels measured. RESULTS: The mouse ileal tissue pathological score and I-FABP levels, as well as serum I-FABP and I-FABP mRNA levels, were significantly higher in the model group than in the control group. Serum I-FABP, I-FABP mRNA, and IL-6 levels were significantly higher in human neonates with NEC than in the healthy group. Logistic regression and receiver operating curve analyses revealed that I-FABP protein/mRNA and IL-6 levels could be diagnostic biomarkers for NEC. CONCLUSIONS: I-FABP protein/mRNA and IL-6 levels are useful biomarkers of intestinal ischemic injury in neonates with NEC. The combined detection of I-FABP protein/mRNA and IL-6 is recommended rather than using a single biomarker.

Lactate acid level and prognosis of neonatal necrotizing enterocolitis: a retrospective cohort study based on pediatric-specific critical care database.

OBJECTIVE: To investigate the relationship between lactate acid level and hospitalization mortality in neonatal necrotizing enterocolitis (NEC). METHOD: Paediatric-specific critical care database collected clinical data from the intensive care unit of Children's Hospital Affiliated to Zhejiang University Medical College from 2010 to 2018. Clinical and laboratory examination information of NEC patients was collected and divided into the death group and discharge group to find out the risk factors affecting the prognosis through univariate and multivariate analysis. RESULTS: Among 104 NEC neonates, the admission age was 7.5 days and the weight was 2.03 kg. Comparing the death group with the discharge group, there were significant differences in therapeutic regimen, pH, serum albumin, total protein, creatinine and lactate acid. Multivariate and threshold effect analysis showed that lactate acid had a linear correlation with hospital mortality, and newborns who died in the hospital had much higher lactate levels than those who were discharged. The mortality of NEC newborns increased by 40-45% for every 1 mmol/L increase in lactate acid level. CONCLUSIONS: There was a correlation between lactate acid level and hospital mortality in newborns with NEC, and lactate acid level was an important index to evaluate the prognosis of NEC.

Hepatoblastoma with neonatal necrotizing enterocolitis: Two case reports.

We report two children with hepatoblastoma (HB) with a history of neonatal necrotizing enterocolitis (NEC). Case 1 was diagnosed with HB at 5 months of age. Liver enlargement was found during the NEC operation at 3 months of age and then was clinically diagnosed by imaging. After six chemotherapy courses, a partial hepatectomy was performed. Three months after ceasing the chemotherapy, a chest computed tomography scan suggested that distant metastasis of the tumor should be considered, and the lesion was removed. However, 9 months after the operation, alpha-fetoprotein concentrations were increased, and abdominal imaging showed a recurrence of the tumor in situ, resulting in a hepatectomy. Case 2 was diagnosed with NEC shortly after birth and underwent an intestinal resection and anastomosis 1 month later. He was diagnosed with HB at 3 years of age. Hepatectomy was performed after five courses of chemotherapy. Chemotherapy was stopped after 10 courses, and alpha-fetoprotein concentrations were normal. At present, both children have survived and are in a healthy condition. Physicians should be aware of the possibility of HB and a history of NEC in children. Premature birth and low birth weight are common factors leading to the pathogenesis of HB and NEC. The association between these two diseases requires further study.

Comparison and Investigation of Exosomes from Human Amniotic Fluid Stem Cells and Human Breast Milk in Alleviating Neonatal Necrotizing Enterocolitis.

In view of the devastating impact of neonatal necrotizing enterocolitis (NEC) on newborns, the research on its intervention is particularly important. Although exosomes from human amniotic fluid stem cells (AFSC) and human breast milk (HBM) can protect against NEC, their mechanisms remain unclear. Here, we intend to compare the intervention effects of two types of exosomes on NEC mouse model and reveal their respective regulatory mechanisms. In general, both AFSC-derived exosomes (AFSC-exos) and HBM-derived exosomes (HBM- exos) can alleviate NEC- associated intestinal injury, significantly reduce NEC score, and reduce systemic and ileal inflammation and NEC related brain injury during experimental NEC. However, the mode and mechanism of action of the two sources of exosomes were not identical. In vivo, the number of ileal crypts was more significantly restored after HBM-exos intervention than AFSC-exos, and in vitro, HBM-exos preferentially inhibited the inflammatory response of intestinal epithelial cells (IECs), whereas AFSC-exos preferentially regulated the migration of IECs. Mechanistically, GO and KEGG analyses revealed the different therapeutic mechanisms of AFSC-exos and HBM-exos in NEC. Taken together, our results illustrate that AFSC-exos and HBM-exos reduce the severity of experimental NEC and intestinal damage through different mechanisms, supporting the potential of cell-free or breast milk free exosome therapy for NEC.

Paneth cell development in the neonatal gut: pathway regulation, development, and relevance to necrotizing enterocolitis.

Paneth cells (PCs) are intestinal epithelial cells (IECs) that contain eosinophilic granules, which are located in Lieberkühn crypts. An increasing number of animal and human experiments have indicated that PCs are involved in the progression of a variety of intestinal as well as systemic inflammatory responses including necrotizing enterocolitis (NEC). NEC is an enteric acquired disease with high mortality that usually occurs in premature infants and neonates, however the underlying mechanisms remain unclear. In this review, we summarize the features of PCs, including their immune function, association with gut microbiota and intestinal stem cells, and their mechanism of regulating IEC death to explore the possible mechanisms by which PCs affect NEC.

Deciphering the role of TNF­α­induced protein 8­like 2 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.