New Therapeutic Targets for Neonatal Necrotizing Enterocolitis: Intestinal FXR and Ferroptosis

Necrotizing Enterocolitis (NEC) is a common and often fatal intestinal disease in preterm infants, yet its mechanisms remain incompletely understood. A recent study published in Immunity has unveiled the molecular mechanisms by which the bile acid receptor FXR exacerbates NEC through regulating intestinal epithelial cell ferroptosis and ILC3 dysfunction, and has proposed potential therapeutic targets. This article will guide you through the key technologies, experimental designs, and core conclusions of this study.

I. Comprehensive Core Experimental Technologies

This study integrates multi-omics technologies with cutting-edge molecular biology methods to validate scientific hypotheses step by step:

1. Single-cell RNA sequencing (scRNA-seq)

Analyzing the transcriptional heterogeneity of intestinal epithelial cells (IECs) in NEC mice to identify the specific high expression of FXR in enterocytes.

2. Flow Cytometry and Sorting

Detecting FXR protein expression in IECs and sorting ILC3 (CD45⁺Lin⁻CD90.2⁺CD127⁺KLRG1⁻) for functional analysis.

3. Multiplex Immunohistochemistry (mIHC)

Panel design:

FGF19 localization: FGF19 (red) + EpCAM (white, marking epithelial cells) + DAPI (blue, nuclear staining).

FXR and lipid peroxidation: FXR (red) + 4-HNE (green, lipid peroxidation marker) + EpCAM (white) + DAPI (blue).

Colocalization analysis reveals the spatiotemporal association between FXR and oxidative damage in the intestines of NEC patients.

4. Lipidomics (LC-MS/MS)

Quantifying phospholipid peroxides (PE-PUFAs) to confirm that FXR promotes ferroptosis by upregulating ACSL4.

5. Gene Editing and Animal Models

Constructing intestinal-specific FXR knockout mice (Fxr^AlEC) combined with an NEC induction model (hypoxia + low temperature + formula feeding).

6. Chromatin Immunoprecipitation (ChIP) and Luciferase Reporter

Verifying that FXR directly binds to the ACSL4 promoter to regulate its transcriptional activity.

II. Sample Grouping Logic

The study validates through both clinical cohorts and animal models:

1. Clinical Samples

NEC group: 6 NEC-confirmed infants (plasma, intestinal tissue).

Control group: 6 age-matched non-NEC infants (normal intestinal segments taken during congenital intestinal malformation surgery).

2. Mouse Experiments

Genotype grouping: Fxr^AlEC (intestinal FXR knockout) vs. Fxr^fl/fl (wild-type control).

Intervention grouping:

FXR modulation: Agonist Fexaramine, antagonist Gly-β-MCA.

Ferroptosis intervention: Inhibitor Liproxstatin-1, ACSL4 inhibitor PRGL493.

Microbiota intervention: Fecal microbiota transplantation from NEC patients + butyrate supplementation.

III. Groundbreaking Conclusions

1. FXR as a Key Driver of NEC

FXR expression is significantly elevated in the intestines of NEC patients and positively correlated with plasma FGF19 (an FXR downstream target), indicating that FXR activation exacerbates the disease.

2. Dysbiosis of Microbiota-Epithelial Cell Interaction

NEC-associated dysbiosis reduces short-chain fatty acids (SCFAs, such as butyrate), lifting their inhibitory effect on FXR and leading to enterocyte ferroptosis.

3. Ferroptosis-ILC3 Axis Worsening Inflammation

FXR transcriptionally activates ACSL4, promoting lipid peroxidation and ferroptosis; oxidized phospholipids (PEox) released by dying epithelial cells inhibit ILC3 secretion of IL-22, weakening intestinal barrier repair.

4. New Therapeutic Strategies

Targeted inhibition of intestinal FXR, ACSL4, or ferroptosis significantly alleviates NEC symptoms in mice, increasing survival rates by over 50%.

IV. Clinical Translation Implications

1. Diagnostic Biomarkers: Plasma FGF19 and lipid peroxidation products (LPO) may serve as early warning indicators for NEC.

2. Therapeutic Directions: Developing intestinal-specific FXR antagonists (e.g., Gly-β-MCA) or ferroptosis inhibitors, or modulating the microbiota through probiotics/butyrate.

Summary

This study is the first to reveal the central role of the FXR-ferroptosis-ILC3 axis in NEC, providing a new perspective for understanding the molecular mechanisms of intestinal inflammation and offering precise targets for clinical intervention. The integration of multi-omics technologies and cross-species models highlights the strong power of translational medical research.

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