Abstract: Objective: To investigate the effects of ginseng-derived extracellular vesicles (Gin-DEVs) on radiation-induced polarization and inflammatory responses in RAW264.7 macrophages and to explore the underlying mechanisms. Methods: Gin-DEVs were extracted and purified from fresh ginseng using ultracentrifugation combined with sucrose density gradient centrifugation. Their morphology, particle size, and zeta potential were characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The effects of different concentrations of Gin-DEVs on the viability of RAW264.7 macrophages were assessed using the CCK-8 assay to determine the optimal concentration for subsequent experiments. The uptake of Gin-DEVs by RAW264.7 cells was observed using fluorescence tracing. A radiation injury model was established by exposing cells to 6 Gy γ-ray irradiation. RAW264.7 cells were divided into a blank control group, a model group, and a Gin-DEVs intervention group. The proportions of M1-type (CD80 +CD206–) and M2-type (CD80+CD206+) macrophages were determined by flow cytometry. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-10, and nitric oxide (NO) in the cell culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Intracellular reactive oxygen species (ROS) levels were detected using a fluorescent probe, 2’,7’- dichlorodihydrofluorescein diacetate (DCFH-DA). The protein expression levels of stimulator of interferon genes (STING) and its downstream factors, TANK-binding kinase 1 (TBK1), phosphorylated TBK1 (p-TBK1), interferon regulatory factor 3 (IRF3), and phosphorylated IRF3 (p-IRF3), were analyzed by Western blot. Results: The purified Gin-DEVs exhibited a typical bilayer membrane structure, with an average particle size of (155.67 ± 0.38) nm and a particle concentration of 1.0 × 10 12 particles/mL. CCK-8 results indicated that Gin-DEVs promoted the proliferation of RAW264.7 cells in a concentration-dependent manner. Flow cytometry results showed that Gin-DEVs significantly inhibited radiation-induced M1 macrophage polarization and promoted M2 polarization (P < 0.05). ELISA results demonstrated that compared with the control group, Gin-DEVs significantly reduced the levels of TNF-α, IL-1β, and NO but increased the level of IL-10 (P < 0.01). DCFH-DA fluorescent probe results revealed that Gin-DEVs significantly suppressed radiation-induced intracellular ROS accumulation (P < 0.01). Western blot analysis revealed that compared with the model group, Gin-DEVs significantly suppressed the protein expression of STING, p-TBK1, and p-IRF3 (P < 0.05). Conclusion: Gin-DEVs may regulate radiation-induced macrophage polarization and suppress inflammatory responses and oxidative stress by inhibiting the activation of the cyclic GMP-AMP synthase (cGAS)-STING signaling pathway.

Key words: ginseng-derived extracellular vesicles; radiation damage; macrophage polarization; inflammatory response; cyclic GMP-AMP synthase-stimulator of interferon genes pathway