Jun 05, 2026
Protective Effect of Gut Microbiota-Derived Metabolites on Necrotizing Enterocolitis in Preterm Infants
- Rong Chen1,
- Xiaojun Xiaojun Lin2,
- Wenlong Xiu2
- 1Department of Neonatology, Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University, Fuzhou, China;
- 2College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics , Fujian Medical University, Fuzhou, China
Protocol Citation: Rong Chen, Xiaojun Xiaojun Lin, Wenlong Xiu 2026. Protective Effect of Gut Microbiota-Derived Metabolites on Necrotizing Enterocolitis in Preterm Infants. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp4e7jgzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 03, 2026
Last Modified: June 05, 2026
Protocol Integer ID: 318485
Keywords: protective role of gut microbiota, protective effect of gut microbiota, metabolites on necrotizing enterocolitis, gut microbiota, gut microbiota dysbiosi, profiling of intestinal metabolite, intestinal metabolite, metabolites in nec development, microbiota dysbiosi, lithocholic acid on the intestine, necrotising enterocolitis, necrotizing enterocolitis, protective effects of niacin, intestine, severe inflammatory disorder of the intestine, mortality in preterm infant, gut, derived metabolite, preterm infant, niacin, nec development, risk factor for nec, preterm infants at onset
Funders Acknowledgements:
Natural Science Foundation of Fujian Province
Grant ID: Grant No. 2022J02049
Fujian Provincial Health Technology Project
Grant ID: Grant No. 2024ZD01005
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Abstract
Necrotising enterocolitis (NEC) is a severe inflammatory disorder of the intestines that remains a leading cause of morbidity and mortality in preterm infants. Given that gut microbiota dysbiosis is a recognized risk factor for NEC, this study investigates the potential intestinal-protective role of gut microbiota-derived metabolites in NEC development.
This research project comprises two major components:
- Profiling of intestinal metabolites in preterm infants at onset and post-treatment based on necrotizing enterocolitis (NEC) stage.
- Protective effects of niacin and lithocholic acid on the intestine in NEC.
Materials
Fecal samples, isotope-labeled internal standards, Vanquish ultra-high performance liquid chromatograph, Waters ACQUITY UPLC BEH Amide liquid chromatography column, Orbitrap Exploris 120 mass spectrometer, Xcalibur software (version 4.4, Thermo), ProteoWizard software, R package, BiotreeDB (V3.0) database, Waters ACQUITY UPLC BEH C18 liquid chromatography column, BeNa Culture Collection (BNCC, China), Dulbecco's Modification of Eagle's Medium (DMEM, Hyclone), fetal bovine serum (FBS, Gibco), trypsin digestion (Gibco), nicotinic acid (NA) (Sigma-Aldrich), lithocholic acid (LCA) (OriLeaf), LPS (Sigma-Aldrich), CCK-8 working solution (Invitrogen), 4% paraformaldehyde, automatic tissue dehydrator (Kuohai Medical), anti-ZO-1 antibody (Sanying), anti-occludin antibody (Sanying), FITC-coupled secondary antibody (Sanying), DAPI, TRIzol kit (Invitrogen), chloroform, isopropanol, 75% anhydrous ethanol (prepared with DEPC water), Nanodrop spectrophotometer (Thermo Fisher Scientific, USA), PrimeScript RT Reagent kit (TaKaRa), ultrasonic processor, bicinchoninic acid (BCA) assay, Interleukin-1β (IL-1β, Abclonal), IL-6.
Non-Target Metabolomics Analysis and High-Throughput Targeting of Bile Acids
The P-value of the t-test and the variable importance in projection (VIP) of the first principal component of the OPLS-DA model. Metabolites were considered significantly altered when VIP 3e 1 and P 3c 0.05. Enrichment analysis was subsequently performed on the metabolic pathways mapped to the differentially expressed metabolites using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The Differential Abundance Score (DA Score) was then calculated to quantify pathway-level alterations. Subsequent pathway analysis was conducted using the MetaboAnalyst (V5.0) website, and the results of the enrichment analysis and topological analysis were integrated to identify the metabolic processes most closely related to the observed differences in metabolites. MetOrigin was utilized to conduct a traceability analysis of metabolites and discern their provenance.
Cell culture
The rat small intestinal crypt epithelial cell line (IEC-6) was obtained from BeNa Culture Collection (BNCC, China). Cells were cultured in Dulbecco's Modification of Eagle's Medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco) in an incubator at 37°C in a 5% CO2 incubator. Passages were conducted through trypsin digestion (Gibco) upon reaching 80% cell confluence.
Cell proliferation assay
Cells (104 cells/well) were cultured in 96-well plates. After 24 h, the medium was replaced. The cells were then treated with different doses of nicotinic acid (NA) (Sigma-Aldrich), lithocholic acid (LCA) (OriLeaf), and LPS (Sigma-Aldrich) for 24 h. A 10 μL CCK-8 working solution (Invitrogen) was then added, and after 1-h incubation period, the optical density values were assessed at 450 nm to determine cell viability. Cell viability was quantified using the formula:
Cell viability (%) = (optical density (OD)treated sample – ODblank)/(ODcontrol sample – ODblank) × 100%
Establishing the Rat NEC model and treatment
To ensure animal welfare, endeavours were made to minimize both the number of animals used and their discomfort. The sample size was determined on the basis of the survival rates observed in the modelling experiments and with reference to similar studies [8,9]. All experimental procedures were approved by the Ethics Committee of Fujian Provincial Maternity and Child Health Hospital(AEC-SFY-XM2025-40).
Ten pregnant SPF-grade SD rats were acquired from Sibeifu Biotechnology Co., Ltd. (Beijing, China) and housed in an SPF-grade animal facility. After the dams gave birth naturally, 70 3-day-old newborn pups were randomly assigned to four groups (control: n=10; NEC: n=20; NEC+NA: n=20; NEC+LCA: n=20) without sex stratification, with body weights ranging approximately from 8 to 11 g. The random allocation sequence was generated by a researcher independent from the endpoint measurements. During the experimental period, the newborn rats in the control group were co-housed with their dams and breastfed. The pups assigned to remaining groups were housed in an incubator (36 ± 1 °C, 45–60 % humidity) and received formula milk via gavage.
The NEC modeling protocol involved thrice-daily cycles of formula feeding (30–50 μL g−1, five feeds per 24 h), hypoxia (4 % O2 / 95 % N2, 10 min), and cold stress (4 °C, 10 min) for three consecutive days, plus a single daily intragastric dose of LPS (5 mg/kg) [14,15]. In the NEC + NA and NEC + LCA groups, NA (50 mg/kg/d) or LCA (0.8 mmol/kg/d), respectively, were incorporated into the first formula feed each morning throughout the three-day modelling period.
All experimenters had received animal ethics training prior to the study initiation. During the 3-day experimental period, neonatal rats in each group were closely monitored for feeding behavior, activity level, abdominal distension, diarrhea, and bloody stools. Survival rate and body weight were recorded daily before the first feeding. All pups were closely monitored for signs of distress throughout the experiment. During the critical colitis phase, supportive care (including milk, diet gel, hydrogel, and/or subcutaneous fluid administration) was provided as needed. Animals exhibiting signs indicative of pain or discomfort, such as lethargy or weight loss greater than 15%, were humanely euthanized immediately. Once the animals met the pre-determined endpoint criteria, they were deeply anesthetized via intraperitoneal injection of avertin (500 mg/kg), followed by euthanasia via decapitation within 30 minutes to minimize suffering. All animal procedures were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee.
The intestinal tract (from the distal end of the duodenum to the rectum) was carefully excised and macroscopically examined for NEC-like pathological features such as hemorrhage, necrosis, or pneumatosis intestinalis. Intestinal contents were gently cleared, and approximately 1 cm segments of ileum (near the ileocecal region) and colon were collected and stored at -80°C for subsequent analysis. To ensure methodological rigor and minimize bias, a blinding procedure was implemented during the outcome assessment phase. The investigators responsible for the histological evaluations and molecular analyses were unaware of the group allocation of the samples throughout the data collection and analysis process.
Hematoxylin-eosin Staining and Immunofluorescence technique
Ileum and colon tissues (1-2cm) near the ileocecal region were collected and immediately fixed with 4% paraformaldehyde. The tissues were placed in an automatic tissue dehydrator (Kuohai Medical) for dehydration, wax dipping, and paraffin embedding. The samples were then cut into 4-μm-thick sections with a slicer. To evaluate intestinal morphology, formalin-fixed, paraffin-embedded sections were stained with haematoxylin and eosin and examined by two blinded investigators under a light microscope; necrotising enterocolitis histological lesion scores were then assigned according to a validated grading system [16].
For ZO-1 or occludin staining, the sections were blocked in 5% bovine serum albumin (BSA, Boster) for 30 min and incubated overnight at 4 °C with an anti-ZO-1 or anti-occludin antibody (Sanying). Then, they were incubated for 1 h at room temperature with a FITC-coupled secondary antibody (Sanying). After staining the nuclei with DAPI, all sections were observed and photographed using a fluorescence microscope (Olympus, Japan).
Reverse transcription real-time quantitative PCR (RT-qPCR)
Total RNA was extracted from the ileum and colon tissue using a TRIzol kit (Invitrogen), followed by extraction, precipitation, and washing steps using chloroform, isopropanol, and 75% anhydrous ethanol (prepared with DEPC water), respectively. RNA quantification was performed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA).
Subsequently, cDNA synthesis was carried out through reverse transcription employing a PrimeScript RT Reagent kit (TaKaRa). The reaction conditions were as follows: denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 15 s, and then annealing at 60°C for 30 s. The data were analyzed using the 2 -ΔΔCt method.
Enzyme Linked Immunosorbent Assay (ELISA)
Ten milligrams of intestinal tissue were weighed accurately, minced finely and homogenised in ice-cold lysis buffer using an ultrasonic processor until total tissue disruption was achieved. After removing the debris by centrifugation, the total protein concentration was determined using the bicinchoninic acid (BCA) assay, and was then adjusted uniformly to 1 mg/mL for all samples. Interleukin-1β (IL-1β, Abclonal), IL-6 were used for the quantification of inflammation following the manufacturer’s protocols.
Statistical Analysis
The statistical analysis was performed using SPSS 26.0 software. The data are expressed as the mean ± standard deviation (SD), the median (interquartile range), or counts (percentages), as appropriate. The data were tested for normality using the Kolmogorov-Smirnov method and for chi-square using the Levene method. Continuous variables were expressed as mean ± SD if normally distributed and compared by two-sample t-test or one-way ANOVA as appropriate; otherwise, they were reported as median (interquartile range) and analyzed with non-parametric tests. Categorical variables were tested using the chi-square test or the Fisher's exact test. The significance level for all tests was set at Pc0.05. GraphPad Prism 8.4 software was used for image visualization and graphing. All experiments were performed with randomization and blinding.
For metabolome data analysis, specific matched statistics methods were used, as mentioned above.
Protocol references
[12] MetaboAnalyst (V5.0) website
[13] MetOrigin
[8] Similar studies
[9] Similar studies
[14] Similar studies
[15] Similar studies
[16] Validated grading system