Bone marrow mesenchymal stem cell-derived exosomes improve pyroptosis and mitochondrial integrity through miR-515-5p-mediated TLR4/NLRP3/GSDMD axis in rheumatoid arthritis.

BACKGROUND: Bone marrow mesenchymal stem cell (BMSC) therapy can significantly improve the outcomes of rheumatoid arthritis (RA). This study explores the protective role of BMSC-derived exosomes (BMSCs-Exos) in RA through modulation of pyroptosis and mitochondrial integrity via the microRNA (miR)-515-5p/Toll-like receptor 4 (TLR4)/NOD-like receptor protein 3 (NLRP3)/gasdermin D (GSDMD) pathway. METHODS: Exosomes were isolated from rat BMSCs, with or without miR-515-5p transfection. Exosomes were identified and analyzed through transmission electron microscopy, tunable resistive pulse sensing, and protein profiling via Western blot analysis. An in vitro RA model was established by stimulating RA fibroblast-like synoviocytes (RA-FLSs) with interleukin-1β (IL-1β). Co-culture of RA-FLSs with miR-515-5p-enriched BMSCs-Exos was used to evaluate inflammation, extracellular matrix (ECM) adhesion, migration, and invasion. Dual-luciferase reporter and RNA immunoprecipitation assays were performed to validate the targeting relationship between miR-515-5p and TLR4. Pyroptosis, reactive oxygen species (ROS) generation, and mitochondrial function were assessed. In vivo effects were confirmed using the collagen-induced arthritis (CIA) rat model. RESULTS: In RA-FLSs, BMSCs-Exos suppressed ECM adhesion, migration, and invasion, and attenuated IL-1β-induced inflammation through the TLR4/NLRP3/GSDMD pathway. BMSCs-Exos inhibited pyroptosis and improved mitochondrial function. Inhibition of miR-515-5p reduced cell viability, caused morphological changes, elevated cytosolic calcium (Ca²⁺), and increased mitochondrial ROS, activating caspase-dependent apoptosis and TLR4/NLRP3/GSDMD-mediated pyroptosis. In CIA rats, BMSCs-Exo treatment significantly alleviated joint damage, reduced pro-inflammatory cytokines, and protected against bone erosion. CONCLUSION: BMSCs-Exos ameliorate RA progression by secreting miR-515-5p, which targets the TLR4/NLRP3/GSDMD pathway, thereby inhibiting pyroptosis and preserving mitochondrial homeostasis in RA-FLSs.