Enterococcus faecium MMX derived from traditional fermented mare's milk alleviates colitis by modulating the LCN2-NLRP3 pyroptosis axis.

Ulcerative colitis (UC) is a relapsing inflammatory disorder of the intestine for which safe and effective therapeutic strategies are urgently needed. The present study aimed to isolate a novel probiotic strain from traditionally fermented mare's milk (koumiss) and to characterise its molecular mechanism of action in alleviating UC through modulation of lipocalin-2 (LCN2) and the NLRP3 inflammasome-mediated pyroptosis pathway. We conducted a systematic investigation integrating microbiological characterisation, whole-genome sequencing, bioinformatic mining and a DSS-induced mouse model of colitis. The results suggested that the isolated Enterococcus faecium MMX strain exhibited no haemolytic activity, strong bile salt tolerance, absence of typical virulence factors, and carried certain resistance genes, while also harbouring multiple probiotic-associated functional genes. Multi-dataset machine learning analysis identified LCN2 as a key biomarker of UC, with its expression highly positively correlated with neutrophil infiltration. In vivo experiments revealed that E. faecium MMX significantly improved the disease activity index, restored the intestinal epithelial barrier (with recovery of E-cadherin expression), promoted goblet cell regeneration and markedly suppressed neutrophil infiltration. Mechanistically, E. faecium MMX treatment was associated with reduced total LCN2 expression in colonic tissue and a shift in its cellular source from a predominantly pro-inflammatory neutrophil-derived localisation to an epithelial cell-derived localisation, accompanied by suppression of the NF-κB pathway (as evidenced by reduced p-p65 levels), which correlated with inhibition of the NLRP3/cleaved-caspase-1/GSDMD-N/IL-1β pyroptosis signalling axis and diminishing the release of pro-inflammatory cytokines, including mature IL-1β and mature IL-18, as confirmed by ELISA and western blot analyses. Collectively, these findings suggest that E. faecium MMX may exert its anti-colitic effects by remodelling the host immune microenvironment, downregulating LCN2 and NF-κB signalling, and inhibiting pyroptosis, positioning it as a promising candidate for microbiota-based intervention in UC.