AI-2 combined with Lactobacillus rhamnosus GG remodels gut microbiota structure to alleviate intestinal oxidative stress injury in a mouse necrotizing enterocolitis model

Necrotizing enterocolitis (NEC) is a devastating intestinal disease primarily affecting preterm infants. This study aimed to explore the efficacy of Lactobacillus rhamnosus GG (LGG) combined with quorum-sensing molecule autoinducer-2 (AI-2) in a neonatal mouse model of NEC. NEC was induced in neonatal mice, which were then randomly assigned to the NEC or treatment groups (NEC + AI-2, NEC + LGG, and NEC + LGG + AI-2), with uninduced mice as control group. Disease severity, intestinal barrier integrity, inflammatory responses, scanning electron microscopy (SEM), gut microbiota structure, transcriptomic profiling, and oxidative stress markers were comprehensively evaluated. The LGG + AI-2 co-treatment demonstrated the most effective protection against NEC. It markedly alleviated clinical symptoms and intestinal histopathological damage, with additive benefits compared with LGG or AI-2 monotherapy. Mechanistically, the combination enhanced the intestinal barrier by upregulating the tight junction protein zona occludens-1 (ZO-1), reducing the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and increasing the levels of interleukin-10 (IL-10) by inhibiting nuclear factor-kappa B (NF-κB) activation. SEM results indicated that LGG combined with AI-2 restored intestinal biofilm formation and colonization of beneficial commensal bacteria. Gut microbiota analysis revealed that LGG + AI-2 ameliorated microbial balance, increasing diversity and selectively enriching beneficial bacteria such as Clostridium butyricum while suppressing the growth of pathogens such as Escherichia coli. Transcriptomic analysis identified 131 core differentially expressed genes, predominantly enriched in glutathione metabolism and oxidative stress pathways. Accordingly, the combination treatment rescued redox homeostasis, evidenced by increased reduced glutathione (GSH) levels, decreased malondialdehyde (MDA) and oxidized glutathione (GSSG) contents, as well as restored expression levels of the key antioxidant regulators glutathione peroxidase 4 (GPX4) and nuclear factor erythroid 2-related factor 2 (NRF2). The combination of LGG and AI-2 confers a potent protective effect against NEC by simultaneously improving intestinal integrity, modulating inflammation and microbiota, and alleviating oxidative stress, highlighting a promising novel combined therapeutic strategy for NEC.