Abstract BACKGROUND Cuprotosis, a copper-dependent regulated cell death pathway, has been increasingly linked to inflammatory disorders, yet its contribution to ulcerative colitis (UC) pathogenesis remains poorly defined. Preliminary evidence suggests that excessive copper accumulation in epithelial cells may disrupt barrier function and amplify oxidative injury, highlighting the need to clarify its mechanistic involvement in UC. Aims The present study aimed to establish the occurrence of epithelial cuprotosis in experimental colitis and to determine whether extracellular vesicles (EVs) derived from Lactobacillus johnsonii exert protective effects by modulating host copper metabolism. METHODS Experimental colitis was induced in BALB/c mice by dextran sulfate sodium (DSS) administration. Animals received oral supplementation with L. johnsonii-derived EVs, and colonic tissues were analyzed for copper accumulation, epithelial barrier integrity, and expression of cuprotosis-associated markers. Complementary in vitro assays were conducted in inflamed NCM460 and Caco-2 cells to assess cytokine release, oxidative stress, barrier-related genes, and cuprotosis proteins. Protein interactions between GroL (a dominant EV protein) and the copper importer SLC31A1 were investigated using co-immunoprecipitation and GST pull-down approaches. RESULTS DSS-induced colitis was characterized by intracellular copper overload, oxidative stress, and dysregulation of cuprotosis-related genes and proteins. Oral delivery of EVs significantly reduced copper accumulation, restored barrier gene expression, and alleviated oxidative injury. In epithelial cell models, EVs suppressed proinflammatory cytokine secretion and normalized cuprotosis marker expression. Mechanistically, GroL, a dominant EV protein, accumulated in the colon, was internalized by epithelial cells, and associated with SLC31A1 in co-immunoprecipitation, accompanied by reduced intracellular copper and attenuated cuprotosis. GST pull-down showed no direct binding, suggesting complex-dependent interactions requiring native membrane contexts. CONCLUSIONS These results identify cuprotosis as a previously unrecognized feature of UC epithelial injury and demonstrate that GroL-enriched EVs mitigate copper-mediated cytotoxicity by regulating host copper transport. This work reveals a novel microbiota–host protein interaction axis with potential therapeutic implications for inflammatory bowel disease.
Zhao et al. (Thu,) studied this question.