hnRNPU regulates the sorting of exo-miR-125a-5p in colon epithelial cells and its role in realgar-induced central nervous system toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Elucidating the mechanisms underlying realgar-induced toxicity is essential for the early prevention and treatment of arsenic-related adverse effects and for reducing the associated healthcare burden.

AIM OF STUDY: Owing to its poor solubility, realgar preferentially accumulates in the intestine after oral administration. This study aimed to elucidate the molecular mechanism by which realgar regulates exosomal miR-125a-5p (exo-miR-125a-5p) sorting in colonic epithelial cells and to investigate the role of exo-miR-125a-5p in realgar-induced central nervous system (CNS) toxicity.

MATERIALS AND

METHODS

A chronic realgar-exposed mouse model was established and combined with AAV9-mediated hnRNPU knockdown, miR-125a-5p-enriched exosome administration, and probiotic intervention. Complementary in vitro studies were performed using YAMC cells subjected to hnRNPU silencing or co-treatment with realgar and L-cysteine sulfinic acid (LCSA). Small RNA sequencing, 16S rDNA sequencing, quantitative proteomics, untargeted metabolomics, RNA immunoprecipitation (RIP), miRNA pull-down, fluorescence in situ hybridization (FISH), Western blotting, and other molecular biological approaches were integrated to systematically investigate the molecular mechanism underlying realgar-regulated exo-miR-125a-5p sorting in colonic epithelial cells and its contribution to realgar-induced CNS toxicity.

RESULTS

Realgar exposure induced dose-dependent colonic injury, altered the exosomal miRNA profile of colonic epithelial cells, and significantly reduced the secretion of exo-miR-125a-5p. Mechanistically, the RNA-binding protein hnRNPU directly bound mature miR-125a-5p, enhanced its post-transcriptional stability, and promoted its nuclear retention, thereby limiting its loading into exosomes. Realgar-induced gut microbiota dysbiosis reduced the abundance of Lactobacillus and the level of its metabolite, LCSA, resulting in aberrant upregulation of hnRNPU. The consequent reduction in circulating exo-miR-125a-5p relieved repression of the STAT1 signaling pathway, leading to activation of the microglial STAT1-NLRP3 inflammasome axis and exacerbation of realgar-induced CNS toxicity.

CONCLUSION

Realgar induces both colonic and central nervous system toxicity. Mechanistically, realgar disrupts gut microbial homeostasis, depleting Lactobacillus-derived LCSA, which promotes hnRNPU expression and enhances the nuclear retention of mature miR-125a-5p. This process suppresses miR-125a-5p sorting into exosomes, reduces circulating exo-miR-125a-5p levels, and consequently activates the microglial STAT1-NLRP3 inflammasome pathway, thereby aggravating CNS toxicity. These findings identify a previously unrecognized gut microbiota-metabolite-RNA-binding protein axis governing exosomal miRNA sorting and establish exo-miR-125a-5p as a critical mediator linking intestinal injury to neuroinflammation. This study provides new mechanistic insights into realgar-induced colonic and CNS toxicity and expands our understanding of gut-brain axis-mediated toxicological mechanisms.