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.

NLRP3 activation in macrophages promotes acute intestinal injury in neonatal necrotizing enterocolitis.

BACKGROUND: Macrophages are involved in various immune inflammatory disease conditions. This study aimed to investigate the role and mechanism of macrophages in regulating acute intestinal injury in neonatal necrotizing enterocolitis (NEC). METHODS: CD68, nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3), cysteine aspartate-specific protease-1 (caspase-1), and interleukin-1ß (IL-1ß) in paraffin sections of intestinal tissues from NEC and control patients were detected with immunohistochemistry, immunofluorescence, and western blot. Hypertonic pet milk, hypoxia and cold stimulation were used to establish a mouse (wild type and Nlrp3-/-) model of NEC. The mouse macrophage (RAW 264.7) and rat intestinal epithelial cell-6 lines were also cultured followed by various treatments. Macrophages, intestinal epithelial cell injuries, and IL-1ß release were determined. RESULTS: Compared to the gut "healthy" patients, the intestinal lamina propria of NEC patients had high macrophage infiltration and high NLRP3, caspase-1, and IL-1ß levels. Furthermore, in vivo, the survival rate of Nlrp3-/- NEC mice was dramatically improved, the proportion of intestinal macrophages was reduced, and intestinal injury was decreased compared to those of wild-type NEC mice. NLRP3, caspase-1, and IL-1ß derived from macrophages or supernatant from cocultures of macrophages and intestinal epithelial cells also caused intestinal epithelial cell injuries. CONCLUSIONS: Macrophage activation may be essential for NEC development. NLRP3/caspase-1/IL-1ß cellular signals derived from macrophages may be the underlying mechanism of NEC development, and all these may be therapeutic targets for developing treatments for NEC.

The relationship between resilience, anxiety, and depression in Chinese collegiate athletes.

Anxiety disorder is becoming increasingly prevalent across college campuses, particularly among collegiate athletes who face different stressors and challenges than their non-athlete peers. Anxiety and depression are well documented comorbidities. Furthermore, the number of Chinese collegiate athletes suffering from anxiety and depression is increasing. Psychologists have recently begun to take a more prophylactic approach to defend against these disorders through resilience education. Resilience education focuses on developing greater capacity to cope when mental and emotional challenges arise. The purpose of this study was to analyze the relationship between resilience, depression, and anxiety in Chinese collegiate athletes. This study provides additional evidence of the value of incorporating an effective resilience educational module amongst collegiate athletes. Research design: The study employed a survey research design to explore the complex relationship between depression, anxiety, and resilience for collegiate student athletes. Data analysis strategy: Structural Equation Modeling (SEM) was employed to account for any measurement error between the observed items (responses on the instruments) and the latent variables (anxiety, depression, and resilience). The theoretical hypothesized relationship for this study is an inverse relationship between anxiety and depression, on the one hand, and resilience on the other hand. The SEM statistical analysis from this study supported this theoretical model. Effective sample size: 599 collegiate athletes from six different universities in the province of Guangdong, China P.R. participated in this study.

[Clinical analysis of annular pancreas in neonates].

OBJECTIVE: To analyze clinical manifestations, diagnosis and treatment of annular pancreas in neonates. METHODS: Clinical data of 114 neonates with annular pancreas admitted in the Children's Hospital of Zhejiang University from January 2009 to December 2018 were reviewed. The demographic parameters (gestational age, birth weight), clinical manifestations, onset time, results of antenatal examination, associated anomalies, radiological findings, operations, postoperative complications were analyzed. RESULTS: One hundred and two cases were examined by abdominal echography, in which 68 cases showed duodenal obstruction, 4 cases showed annular pancreas. Plain abdomen X-ray examination performed in 113 cases before operation, 76 cases presented double-bubble sign, 12 cases presented single-bubble sign and 5 cases had high-position intestinal obstruction. Upper gastrointestinal radiography (UGI) was performed in 103 cases, which suggested duodenal obstruction in 102 cases. Operations were performed in all cases, of which 69 cases were operated under laparoscopy including 1 case converted to open laparotomy. The mean fasting time after surgery was (7.8±2.7) d, and the mean length of hospital stay was (16.9±10.1) d. Five patients had postoperative complications. The incidence of postoperative complications in antenatal abnormal group was lower than that in the antenatal non-abnormal group (P<0.05); the average fasting time in laparoendscopic surgery group was shorter than that in traditional laparotomy group (P<0.05). CONCLUSIONS: Neonates with recurrent vomiting early after birth should be highly suspected to have annular pancreas. The fetal chromosome examination should be performed with abnormal antenatal screening. Surgery is the only effective way to diagnose and treat annular pancreas, and laparoscopic surgery could be the first choice for experienced doctors.

[Therapeutic experience of type â ¢-b congenital intestinal atresia].

OBJECTIVE: To summarize the clinical characteristics and treatment of type Ⅲ-b congenital intestinal atresia (CIA). METHODS: The clinical data of 12 type Ⅲ-b CIA treated in the Children's Hospital of Zhejiang University School of Medicine from January 2015 to December 2017 were analyzed retrospectively. RESULTS: Of the 12 patients diagnosed as type Ⅲ-b CIA in operation, treatment was refused during operation by their parents in 2 cases. For one child, only the proximal intestine was partly resected in the first operation, dilatation and dysplasia of the duodenum was diagnosed and total duodenum was resected and sutured in the second operation, as the child had postoperative intestinal obstruction. For one child, due to the long distal normal intestine, distal apple-peel like intestine was partly resected without mesenteric reformation. For the rest 8 children total duodenum resection and mesenteric reformation were performed. During the postoperative follow-up, one case was early rejected for further treatment by parents, one case died from complex congenital heart disease, 5 cases had the complication of short bowel syndrome. All 8 survival children received parenteral nutrition support after operation, 5 of whom received parenteral nutrition support for more than 42 days, and they were followed up for 1-3 years after discharge. The short-time efficacy was satisfactory. CONCLUSIONS: For children with type Ⅲ-b CIA, the distal apple-peel like intestine should be preserved as much as possible, the mesenteric reformation should be performed and the proximal dilated bowel should be partly resected and sutured. Postoperative nutritional support and early intestinal rehabilitation contribute to the compensation for rest intestines.

[Transumbilical single-site laparoscopic surgery for congenital duodenal obstruction in neonates].

OBJECTIVE: To evaluate the efficacy and safety of transumbilical single-site laparoscopic surgery for congenital duodenal obstruction (CDO) in neonates. METHODS: A retrospective analysis of clinical data of 15 patients with CDO undergoing transumbilical single-site laparoscopic treatment during November 2017 and January 2018 (single-site group), and 20 patients with CDO undergoing conventional three-hole laparoscopic treatment during August 2017 and October 2017 (three-hole group) was performed. All patients were from the Children's Hospital, Zhejiang University School of Medicine. The operation time, time of initial feeding, time of adequate feeding, length of hospital stay after operation and postoperative complications were compared between two groups. RESULTS: The operations were completed in all patients. No patient converted to laparotomy, and no massive hemorrhage was observed during operation. The operation time of single-site group was (90±10) min for patients with duodenal diamond-shaped anastomosis and (81±15) min for patients with Ladd operation, while those of three-hole group were (85±9) min and (72±11) min, respectively. Postoperative initial feeding time of single-site group was (5.0±1.0) d, and that of the three-hole group was (4.8±0.8) d. The adequate feeding time was (9.0±1.2) d in the single-site group, and (9.3±0.8) d in the three-hole group. The length of hospital stay after operation was (11.2±2.5) d in the single-site group, and (11.5±2.8) d in the three-hole group. There was no significant difference in operation time, postoperative initial feeding time, adequate feeding time and length of hospital stay after operation between two groups (all P>0.05). CONCLUSIONS: Transumbilical single-site laparoscopic surgery for CDO in neonates is safe and effective, and the postoperative abdominal scar is more hidden.

[Complications after laparoscopic Ladd operation for intestinal malrotation in neonates].

OBJECTIVE: To analyze complications after laparoscopic Ladd operation for intestinal malrotation, related causes and possible solutions. METHODS: Clinical data of 81 neonates who underwent laparoscopic Ladd operations for intestinal malrotation in the Children's Hospital, Zhejiang University School of Medicine between January 2015 and January 2018 were reviewed. The abdominal complications and findings during operation and reoperation were analyzed. RESULTS: Operations were successfully completed in all patients, and there was no patient converted to open surgery. The annular pancreas in 6 cases and duodenal diaphragm in 4 cases were confirmed during the operation. The recurrent volvulus developed in 3 patients (3.7%), of whom 2 cases were confirmed to have midgut necrosis during open surgery 1 week and 3 months after laparoscopic Ladd operation, and both finally died; 1 case was corrected by second laparoscopic operation. Cecal perforation occurred in 1 patient (1.2%), which was caused by intensive high frequency coagulation of the appendiceal stump. One patient (1.2%) developed chylous ascites and improved after conservative treatment. Adhesive small bowel obstruction was observed in 3 cases (3.7%), and all relieved after conservative treatment. CONCLUSIONS: Laparoscopic Ladd operation for intestinal malrotation in neonates was effective, and the incidence of abdominal complications may be minimized by experienced skills and strict perioperative management.