Circ_0001666 upregulation promotes intestinal epithelial cell fibrosis in pediatric Crohn's disease via the SRSF1/BMP7 axis.

The epithelial-mesenchymal transition (EMT) is closely associated with Crohn's disease (CD) related intestinal fibrosis, a condition whose prevalence is increasing annually among children. Recently, the CD marker gene microarray screening revealed an upregulation of circ_0001666 in the colon tissues of CD patients, but its underlying mechanisms remain unclear. In this study, we explored the molecular mechanism of circ_0001666 in regulating EMT-mediated fibrosis in CD in vitro. The levels of circ_0001666 and EMT-associated proteins were assessed in CD clinical samples, and a CD cell model was established using TGF-ß1 to induce human intestinal epithelial cells (HIECs). Additionally, the expression levels of genes and proteins related to EMT and fibrosis were analyzed by quantitative real-time PCR and western blot, cell migration, and invasion were assessed via wound healing assay and transwell, respectively, and RNA pull-down and RNA immunoprecipitation assays were performed to verify the relationship between SRSF1 and BMP7 or circ_0001666. Circ_0001666 was overexpressed in the intestinal mucosal tissues of CD patients and was positively correlated with EMT. Silencing circ_0001666 inhibited the migration, invasion, EMT, and fibrosis of HIECs induced by TGF-ß1. Mechanistically, circ_0001666 regulated BMP7 expression by interacting with SRSF1. Furthermore, the effects of inhibiting circ_0001666 on HIECs could be partially reversed by overexpressing SRSF1 or silencing BMP7. Collectively, circ_0001666 regulates TGF-ß1-induced HIEC migration, invasion, EMT, and fibrosis. Circ_0001666 also promoted EMT-mediated fibrosis by interacting with SRSF1 to accelerate BMP7 mRNA decay. These findings provide new insights into the pathogenesis of CD and suggest that circ_0001666 might be a potential therapeutic target for CD.

Clinical Characteristics of Pediatric Cases of COVID-19 in Hunan, China: A Retrospective, Multi-Center Case Series.

Objective: Aimed to investigate the epidemiological characteristics, clinical features, treatment, and short-term prognosis of COVID-19 in children. Methods: Retrospective analysis was conducted in 48 children with COVID-19 admitted to 12 hospitals in eight cities in Hunan province, China, from January 26, 2020 to June 30, 2020. Results: Of the 48 cases, Familial clusters were confirmed for 46 children (96%). 16 (33%) were imported from other provinces. There were 11 (23%) asymptomatic cases. only 2 cases (4%) were severe. The most common symptom was fever (n = 20, 42%). Other symptoms included cough (n = 19, 40%), fatigue (n = 8, 17%), and diarrhea (n = 5, 10%). In the early stage, the total peripheral blood leukocytes count increased in 3(6%) cases and the lymphocytes count decreased in 5 (10%) cases. C-reactive protein and procalcitonin were elevated respectively in 3 (6%) cases and 2 (4%) cases. There were abnormal chest CT changes in 22 (46%) children, including 15 (68%) with patchy ground glass opacity, 5 (22%) with consolidation, and 2 (10%) with mixed shadowing. In addition to supportive treatment, antiviral therapy was received by 41 (85%) children, 11 (23%) patients were treated with antibiotics, and 2 (4%) were treated with methylprednisolone and intravenous immunoglobulin. Compared to 2 weeks follow-up, one child developed low fever and headache during the 4 weeks follow-up, 3 (6%) children had runny noses, one of them got mild cough, and 4 (12%) children had elevated white blood cells and lymphocytes. However, LDH and CK increased at 2 weeks and 4 weeks follow-up. 2 weeks follow-up identified normal chest radiographs in 33 (69%) pediatric patients. RT-PCR detection of SARS-CoV-2 was negative in all follow-up patients at 2 and 4 weeks follow-up. All 48 pediatric patients were visited by calling after 1 year of discharge. Conclusions: Most cases of COVID-19 in children in Hunan province were asymptomatic, mild, or moderate. Close family contact was the main route of infection. It appeared that the younger the patient, the less obvious their symptoms. Epidemiological history, nucleic acid test, and chest imaging were important tools for diagnosis in children.

[A multicenter survey of antibiotic use in very and extremely low birth weight infants in Hunan Province].

OBJECTIVE: To investigate the current status of antibiotic use for very and extremely low birth weight (VLBW/ELBW) infants in neonatal intensive care units (NICUs) of Hunan Province. METHODS: The use of antibiotics was investigated in multiple level 3 NICUs of Hunan Province for VLBW and ELBW infants born between January, 2017 and December, 2017. RESULTS: The clinical data of 1 442 VLBW/ELBW infants were collected from 24 NICUs in 2017. The median antibiotic use duration was 17 days (range: 0-86 days), accounting for 53.0% of the total length of hospital stay. The highest duration of antibiotic use was up to 91.4% of the total length of hospital stay, with the lowest at 14.6%. In 16 out of 24 NICUs, the antibiotic use duration was accounted for more than 50.0% of the hospitalization days. There were 113 cases with positive bacterial culture grown in blood or cerebrospinal fluid, making the positive rate of overall bacterial culture as 7.84%. The positive rate of bacterial culture in different NICUs was significantly different from 0% to 14.9%. The common isolated bacterial pathogens Klebsiella pneumoniae was 29 cases (25.7%); Escherichia coli 12 cases (10.6%); Staphylococcus aureus 3 cases (2.7%). The most commonly used antibiotics were third-generation of cephalosporins, accounting for 41.00% of the total antibiotics, followed by penicillins, accounting for 32.10%, and followed by carbapenems, accounting for 13.15%. The proportion of antibiotic use time was negatively correlated with birth weight Z-score and the change in weight Z-score between birth and hospital discharge (rs=-0.095, -0.151 respectively, P<0.01), positively correlated with death/withdrawal of care (rs=0.196, P<0.01). CONCLUSIONS: Antibiotics used for VLBW/ELBW infants in NICUs of Hunan Province are obviously prolonged in many NICUs. The proportion of routine use of third-generation of cephalosporins and carbapenems antibiotics is high among the NICUs.

[Effect of magnesium-free on glucocorticoid receptor expression in primary cultured cortical neurons of fetal rats in vitro].

OBJECTIVE: To study the changes of glucocorticoid receptor (GR) expression in embryonic rat cortical neurons exposed to transient Mg(2+)-free treatment. METHODS: Six days after rat cortical neuronal cultures, two groups were created based on the medium to which were transiently exposed. The control group was exposed to a physiological solution (PS), and the Mg(2+)-free group was exposed to the same medium as the control group except for the removal of magnesium. The expression of GR mRNA and protein was determined by real-time PCR and immunocytochemistry staining 1, 7 and 12 days after transient Mg(2+)-free treatment. RESULTS: Compared to the control group, the Mg(2+)-free group displayed the significantly less accumulated optical density (AOD) of GR immunoreactivity 12 days after transient Mg(2+)-free treatment (p<0.05). On the contrary, GR mRNA expression increased significantly 1 and 7 days after transient Mg(2+)-free treatment in the Mg(2+)-free group (p<0.05). CONCLUSIONS: GR expression is modified following Mg-free-induced injury in cultured developing neurons in rats.

[Effects of neonatal recurrent seizures on glucocorticoid receptor expression in the rat brain].

OBJECTIVE: To investigate the effets of flurothyl-induced neonatal recurrent seizures on glucocorticoid receptor (GR) expression in the rat brain. METHODS: Forty-eight seven-day-old Sprague-Dawley rats were randomly divided into two groups: control and seizure. Seizures were induced by inhalant flurothyl daily for six consecutive days. Brains were sampled on postnatal days 13, 15 and 19. The expression of GR protein in the cerebral cortex was detected by Western blot and immunohistochemical method. RESULTS: The expression of GR in the cerebral cortical plasma protein was significantly lower in the seizure group than in the control group on postnatal day 15. The expression of GR protein in the cerebral cortical nuclear protein decreased significantly in the seizure group compared with that in the control group on postnatal days 15 and 19 (p<0.05). Compared to the control group, the accumulated optical density (AOD) of GR immunoreactivity (IR) decreased significantly in the parietal cortex on postnatal day 13 (p<0.05), the AOD of GR IR decreased significantly in the parietal cortex and the temporal cortex on postnatal day 15 (p<0.05), and the AOD of GR IR decreased significantly in the parietal cortex, temporal cortex and the frontal cortex in the seizure group on postnatal day 19 (p<0.05). CONCLUSIONS: Recurrent seizures in neonatal rats result in abnormal GR expression in the cerebral cortex which might play an important role in short-term brain injury induced by early recurrent seizures.

[Short-term effects of recurrent neonatal seizures on gamma-aminobutyric acid A receptor alpha1 and beta2 subunit expression in the rat brain].

OBJECTIVE: To investigate the short-term effects of flurothyl-induced neonatal recurrent seizures on gamma-aminobutyric acid A receptor (GABAAR) alpha1 and beta2 subunit expression in the rat brain, and to study the relationship between the alterations of GABAAR subunits in the developing brain and seizure-induced brain injury. METHODS: Sixty-four 7-day-old Sprague-Dawley rats were randomly divided into two groups: control and seizure. Seizures were induced by inhalant flurothyl daily for six consecutive days. The expression of GABAAR alpha1 and beta2 subunits protein in the cerebral cortex and the hippocampus were detected by Western blot and immunohistochemistry method 1 and 7 days after recurrent seizures. RESULTS: Compared to the control, the accumulated optical density (AOD) of GABAAR alpha1 subunit immunoreactivity (IR) in the parietal cortex, the CA3-CA4 regions and the dentate gyrus in seizure rats increased significantly 1 day after recurrent seizures (P<0.05). The AOD of GABAAR alpha1 subunit IR in the parietal cortex, the CA1-CA4 regions and the dentate gyrus in seizure rats increased significantly 7 days after recurrent seizures compared with the control (P<0.05). The expression of GABAAR alpha1 subunit in the hippicampus and the cerebral cortex increased significantly in seizure rats compared with that in control rats 1 and 7 days after recurrent seizures. After 7 days of recurrent seizures, the AOD of GABAAR beta2 subunit IR in the CA1-CA2 regions increased significantly in the seizure group compared with that in the control group (P<0.05), but the AOD of GABAAR beta2 subunit IR in the thalamus decreased significantly in the seizure group compared with that in the control group (P<0.05). The expression of GABAAR beta2 subunit protein in the hippocampus increased significantly in the seizure group compared with that in the control group 7 days after recurrent seizures (P<0.05). CONCLUSIONS: Recurrent neonatal seizures may result in the short-term alterations of GABAAR alpha1 and beta2 subunits expression in the cerebral cortex and the hippocampus in rats, suggesting the alterations of GABAAR subunit expression may be related to the developing brain injury following recurrent seizures.