Euonymus alatus-Derived Extracellular Vesicles Show Immunomodulatory Effects in an Inflammatory Thyroid Follicular...

Objectives: To extract and identify extracellular vesicles (EVs) derived from the traditional Chinese herb Euonymus alatus (EA), characterize their molecular profiles using multi-omics analysis, and assess their regulatory effects and potentially associated signaling pathways in an interferon-γ (IFN-γ)-stimulated inflammatory thyroid follicular cell model mimicking key inflammatory features of autoimmune thyroiditis (AIT). Methods: Differential centrifugation combined with density gradient centrifugation was used to purify EA-derived EVs, and the isolates were characterized using a BCA protein assay, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting (WB). Molecular constituents were examined via label-free proteomics and RNA-seq. Meanwhile, Nthy-ori-3-1 cells were exposed to IFN-γ to establish an inflammatory thyroid follicular cell model; these activated cells were subsequently treated with the purified EVs (EV group). The supernatants were collected and the levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) were determined by ELISA. Comparative transcriptomic and proteomic profiling was employed to identify differentially expressed molecules and related signaling pathways between the model and the EV group. Results: EA-derived EVs were successfully isolated and showed typical EV characteristics. Proteomics identified 3607 proteins enriched in metabolic, immune, and antioxidant processes, while RNA-seq generated 72.16 million clean reads and revealed genes enriched in metabolic and immune pathways. In the IFN-γ-stimulated Nthy-ori-3-1 cells, ELISA results showed that EV treatment significantly reduced the secretion of IL-1β and IL-18 compared to the model group. EV intervention resulted in 138 differential proteins (42 upregulated, 96 downregulated) and 262 differential genes (81 upregulated, 181 downregulated). GO analysis revealed significant enrichment in terms related to inflammatory response, immune response, and regulation of oxidative stress. KEGG pathway analysis demonstrated enrichment in the IL-17, NOD-like receptor, and PPAR signaling pathways, as well as ferroptosis, and Wnt pathway (unique for upregulated genes). Conclusions: EA-derived EVs are rich in molecules that regulate metabolic pathways and immune responses. They may ameliorate inflammation in this inflammatory thyroid follicular cell model, with multi-omics analyses revealing correlative enrichment of multiple signaling pathways. These findings provide preliminary experimental evidence and novel candidate targets for AIT treatment.