Extracellular vesicles ameliorate intrauterine adhesion through Nrf2/GPX4-mediated ferroptosis suppression

• L. crispatus NCU-28 supernatant attenuates intrauterine inflammation and fibrosis. • High-purity EVs are isolated and characterised from L. crispatus NCU-28 • Probiotic EVs exhibit anti-inflammatory and anti-fibrotic effects in vivo and in vitro . • The Nrf2/GPX4 signalling axis mediates the protective effects of the EVs. • This study proposes a novel probiotic EV-based therapeutic strategy for intrauterine adhesion. As pivotal functional mediators of probiotics, extracellular vesicles (EVs) play crucial regulatory roles in diverse disease processes. However, the mechanisms by which vaginal probiotic-derived EVs contribute to intrauterine adhesion (IUA) remain poorly understood. To identify EVs derived from Lactobacillus crispatus NCU-28 and elucidate their anti-inflammatory and anti-fibrotic mechanisms. EVs were isolated and purified from vaginal probiotics and systematically evaluated using both an IUA mouse model and an inflammatory cell model. High-throughput sequencing was integrated with multiple molecular biology techniques to assess the anti-inflammatory and anti-fibrotic effects of EVs and to elucidate the underlying molecular mechanisms. An endometrial inflammatory cell model was established, demonstrating that EVs served as the pivotal active components through which L. crispatus NCU-28 exerted anti-inflammatory and anti-fibrotic effects. Following isolation and purification from L. crispatus NCU-28 culture supernatants, EVs were further evaluated in an IUA mouse model. The results demonstrated that EV treatment significantly downregulated fibrotic markers, including α-SMA and TGF-β1; inhibited the release of pro-inflammatory cytokines such as IL-6 and IL-1β; and restored vaginal microbiota homeostasis. Mechanistic investigations revealed that EVs modulated ferroptosis through activation of the Nrf2/GPX4 signalling pathway. Furthermore, reverse validation using a ferroptosis agonist confirmed that EVs inhibited oxidative stress-induced ferroptosis, thereby promoting endometrial repair and suppressing fibrotic progression. This study identifies EVs as central functional components through which L. crispatus NCU-28 exerts anti-inflammatory and anti-fibrotic effects, and it delineates the mechanistic role of EVs in ameliorating IUA. These findings provide a basis for developing innovative probiotic EV-based treatment strategies.