Excised human specimens during the operation, including jejunums and ileums, were obtained following informed consent from all involved parents of the neonates in the Women and Children’s Hospital of Chongqing Medical University in 2020 with approval by the Institutional Review Board of Chongqing Medical University l from July 1, 2020, to July 30, 2022 (IRB, No.: WCHMU2021-052) in accordance with the relevant guidelines and regulations. Written informed consent was obtained for the participants’ parent/legal guardian to participate in the study. The human samples included necrotizing intestinal samples from NEC patients (2 mm3, n = 8) resected during emergency laparotomy. Normal small intestine control samples (2 mm3, n = 6) were collected from infants undergoing surgery for ileal atresia, intestinal intussusception, Hirschsprung’s disease and imperforate anus. The demographics and characteristics of the controls are listed in Table 1. All samples were subjected to the following assessments: Western blotting, ELISA, and immunofluorescence evaluation. The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
Experimental NEC models and drug interventions
The animal experimental protocols conformed to the guidelines for laboratory animal management (NIH publication No. 85–23) and were reviewed and approved by the animal care and use committee at the Women and Children’s Hospital of Chongqing Medical University, approval number (WCHMU2019-038). eNOS knockout (KO) mice (eNOS−/−, C57BL/6 J background) were kindly gifted by Prof. Jingyu Li from Sichuan University. Wild-type C57BL/6 mice were purchased from the Experimental Animal Center at Chongqing Medical University (Dr. Wenli Han). The previously described NEC stress protocol (formula gavage, hypothermia and hypoxia) was applied to five-day-old mouse pups14,15. Briefly, the pups were subjected to formula gavage every 4 h with 75 mL of puppy canine milk replacer (Pet-Ag, Hampshire, USA) and 15 g of Similac 60/40 (Ross Pediatrics, Columbus, OH, USA). Pups staying with the dam without any other treatment were used as controls. The experimental pups were observed, and clinical manifestations were recorded, including abdominal distension, apnea, rectal bleeding, body weight, feeding volume and mortality, in the following experimental period. Experimental NEC was evaluated by macroscopic bowel examination and microscopic assessment of the morphological and histological performance of the terminal ileum. The degree of mucosal injury of the terminal ileum was checked and scored based on a previously described histological scoring protocol4,5. The NEC diagnosis was verified by histological examination. The pups were euthanized using CO2 for intestinal tract harvest 3–5 days after NEC diagnosis. Part of the terminal ileum was subjected to the appropriate subsequent assessments listed below, such as quantitative real-time PCR (qPCR) and Western blotting.
In the PGE2 management study, litters of newborn pups were intraperitoneally injected with either a single dose of dinoprostone (prostaglandin E2, Sigma Aldrich, 300 mg/kg body weight) or vehicle control (0.9% sodium chloride) at an equivalent volume 30 min before NEC stress. In the L-NAME (N-nitro-L-arginine methyl ester hydrochloride, Sigma–Aldrich) management study, L-NAME was dissolved in the formula gavage at a concentration of 1 g/L and administered every 4 h. The flow diagram of experimental management is presented in Fig. 1.
Preparation of intestinal microvascular endothelial cells
Intestinal microvascular endothelial cells (MIMECs) were isolated and cultured as reported previously4,5. When the MIMECs were cultured for 7 days, we obtained almost pure cultures of MIMECs (90% confluence). The MIMECs were subjected to the preliminary dosage selection assay, and the current dosage of TNF-α (30 ng/mL) was selected for 12 h for subsequent experiments. For pharmacological PGE2 management and inhibition of various PGE2 receptors, the culture system was further cultured with the following reagents and final concentrations: stabilized PGE2 analog 16,16-dimethyl PGE2 (dmPGE2, 1 μM, R&D Systems), EP1 inhibitor (10 μM, SC 51322; R&D Systems), EP2 inhibitor (10 μM, PF 04418948; R&D Systems), EP3 inhibitor (10 μM, L-798,106; R&D Systems), and EP4 inhibitor (10 μM, L-161,982, R&D Systems).
ELISA analysis of VEGF
MIMECs were cultured in 24-well plates at 90% confluence. Before PGE2 management, the serum was deprived for 24 h of starvation. The culture supernatant was collected for VEGF concentration measurement using enzyme-linked immunoassay (ELISA) kits (R&D Systems) following the manufacturer’s instructions.
Nitric oxide (NO) assessments
Isolated intestinal segments were homogenized and centrifuged to remove cell debris, and the supernatant was saved for NO analysis. The NO concentrations in the intestinal samples were determined using the modified Griess reaction and the total NO assay kit (Beyotime Institute of Biotechnology, China) as described previously4,5. NOS inhibition was performed by incubation with L-NAME (500 mM, Sigma‒Aldrich).
Cell proliferation detection
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was conducted to assess cell proliferation. MIMECs were seeded in a 96-well plate (500 cells/well) for 24 h, and TNFα (30 ng/mL) was administered for 12 h in RPMI-1640 medium (Gibco, Australia). MTT solution (20 μL; 5 mg/mL in PBS, pH 7.4; Solarbio Life Sciences, Peking, China) and dimethyl sulfoxide (DMSO) (150 μL; Sigma) were then successively incubated within each well with constant shaking. A Thermo Fisher Scientific microplate reader was utilized for the optical absorbance measurement at 490 nm.
PGE2 concentration measurement
MIMEC lysates and terminal ileum samples were collected and homogenized for protein concentration measurement. Samples were diluted 1:10 with assay buffer before analysis. The concentration of PGE2 in ileal tissue homogenates was determined utilizing a high-sensitivity PGE2 enzyme immunoassay kit (Assay Designs, Ann Arbor, MI, USA) following the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction (RT‒PCR)
MIMEC lysates and terminal ileum segments were collected and subjected to RNA extraction with TRIzol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was then conducted for cDNA using a reverse transcription kit (Roche, Germany). RT‒PCR was performed using a Roche LightCycler 480 real-time PCR instrument (Roche, Germany). The primer sequences for EP1, EP2, EP3, EP4, and eNOS are presented in Table 2. The mRNA expression levels of EP1, EP2, EP3, EP4, IL-6, TNFα, CD31, VEGFR2 and eNOS were normalized to 18S rRNA expression. The annealing temperature was 59 °C for all genes examined.
Intestinal SIgA and β-defensin-2 measurement
PBS containing 0.02% sodium azide was subjected to passage through the intestinal lumen, and then intestinal mucus was obtained. The washout mixture was prepared as previously described (n = 6 in each group)3. The levels of secretory immunoglobulin A (SIgA) and β-defensin-2 in the intestinal mucus were determined using ELISA kits (USCN, China) according to the manufacturer’s instructions.
Western blot analysis
The intestinal samples were processed as previously reported. The total protein concentration was quantified using the Bradford protein assay. The different samples (30 μg protein) were first subjected to electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes for primary antibody incubation. The primary antibodies utilized in the current research included p-eNOSSer1177 (Cell Signaling Technology, USA), eNOS (Cell Signaling Technology), CD31 (Cell Signaling Technology), VEGFR2 (Cell Signaling Technology) and GAPDH (Santa Cruz Biotechnology). The appropriate secondary antibodies were incubated to visualize the immunoreactive bands. β-actin (Sigma) was used as a loading control. The relative intensities of the target bands were measured utilizing Kodak 1D 3.5.4 software (Kodak Scientific Imaging System, Rockville, MD, USA).
Confocal immunofluorescence assays
The slides in different groups were first subjected to incubation with the appropriate primary antibodies, including anti-CD31 (1:50, Sigma) and anti-BrdU (1:50) and then incubated with the appropriate secondary antibodies. DAPI (4’,6-diamidino-2-phenylindole; 1:1,000, Sigma) was used to counterstain the nuclei. Finally, the slides were mounted and analyzed under a fluorescence confocal microscope (Leica Microsystems Heidelberg GmbH, Heidelberg, Germany). Quantitative analysis was performed using the plugin ‘colocalization threshold’ of WCIF ImageJ software (National Institutes of Health, Bethesda, MD, USA) by at least two independent investigators. We selected representative sections in 5 fields for each segment of the intestine (magnification, 200 ×) from 6 mice under each experimental condition.
Bacterial translocation by microbiological culture of blood
Blood from experimental pups was collected in a sterile fashion and plated onto blood agar plates (containing 5% sheep blood) to assess total aerobic bacteria or on BBL Brucella agar plates (containing 5% horse blood; BD) as described previously4,5. The colony-forming units (CFUs) were counted following incubation in aerobic or anaerobic conditions at 37 °C for 48–72 h.
GraphPad Prism (version 6) (GraphPad Software, Inc., La Jolla, CA, USA) was used for data analysis. Continuous parametric data are presented as the mean ± SEM for normal distribution and were analyzed using ANOVA. The survival curve was generated with Kaplan‒Meier analysis following the log-rank (Mantel‒Cox) test. P values < 0.05 were considered statistically significant.
All experiments were approved by the animal care and use committee of Chongqing Medical University.