# Controlled release of coated antioxidants inhibits Citrobacter rodentium colonization in the colon of rats by reducing gut redox potential

## TL;DR

A controlled release system of coated antioxidants reduces gut redox potential, which inhibits harmful bacteria like Citrobacter rodentium and promotes beneficial gut microbes in rats.

## Contribution

A novel controlled release system is introduced to modulate gut redox potential and inhibit pathogen colonization through microbiota reprogramming.

## Key findings

- Coated ferulic acid (FA) more effectively reduced gut redox potential and alleviated Citrobacter rodentium-induced weight loss compared to uncoated FA.
- Coated antioxidants enriched beneficial bacteria like Lactobacillus and suppressed pathogens like Klebsiella.
- Lower redox potential enhanced Muc2 gene expression and inhibited virulence gene expression in C. rodentium.

## Abstract

Intestinal redox potential serves as a critical parameter reflecting the dynamic characteristics of the gut microenvironment. To precisely modulate the intestinal redox potential and evaluate its inhibition of pathogenic colonization, this study built a controlled release system and further investigated its role in gut health under a lower redox potential. The results demonstrated that the controlled release formulation significantly reduced fecal redox potential more effectively than uncoated antioxidants. By optimizing the hydrodynamic size and zeta potential of ethoxyquin (EQ) and ferulic acid (FA), the coated FA formulation maintained high efficiency in reducing redox potential and reversed body weight loss induced by pathogenic infection. Both coated EQ (EQC) and FA (FAC) selectively enriched beneficial genera, such as Lactobacillus and Limosilactobacillus, while suppressing opportunistic pathogens like Klebsiella. Notably, coated FA demonstrated enhanced efficacy in alleviating Citrobacter rodentium (C. rodentium)-induced weight loss and reducing pathogens burden compared to uncoated FA. Mechanistically, coated FA promoted the enrichment of Lactobacillus reuteri (L. reuteri), suppressed the proliferation of Enterobacteriaceae, and enhanced intestinal Muc2 gene expression. Functional metagenomic analysis revealed that FAC significantly downregulated ABC transporter activity in Enterobacteriaceae, thereby impairing biofilm formation and synergizing with mucus secretion to inhibit pathogen colonization. Further in vitro co-culture trials confirmed that under a lower redox system, L. reuteri had a stronger inhibitory effect on C. rodentium as well as the expression of their virulence genes ((tir, ler). Collectively, these findings suggest that precise modulation of colonic redox potential through controlled release strategies represents a promising approach to enhance host defense against enteric pathogens via microbiota reprogramming.

Scheme: Graphical scheme of controlled release delivery and its enhancement of colonization resistance against Enterobacteriaceae through modulation of virulence and microbiota function. Following oral administration, the coated antioxidants regulated gut redox potential, thereby reshaping the composition of microbiota in colon. The formulation effectively lowered redox potential, enriched beneficial microbiota (including Akkermansia, Lactobacillus reuteri, and Limosilactobacillus), and enhanced mucus secretion (↑Muc2), thereby strengthening the resistance to pathogens colonization. Mechanistically, this approach inhibited quorum sensing and biofilm formation in enteric pathogens by restricting iron availability and Lactobacillus reuteri suppressed C. rodentium growth and virulence under low redox potential.Image 1