Ozone saline solution polarizes microglial cells towards an anti-inflammatory phenotype

Ozone (O3) therapy has demonstrated antioxidant and anti-inflammatory properties, but the systemic administration of ozonated saline solution (O3SS) remains underexplored. This study evaluates the cytotoxicity, antioxidant response, and immunomodulatory effects of O3SS on murine microglial (BV2) and human endothelial (HUVEC) cells. Cells were exposed to increasing doses of O3 (1, 5, or 10 μg/NmL) dissolved in saline. Viability assays showed that low doses (1 and 5 μg/NmL) enhanced cell proliferation without cytotoxicity, while the highest dose (10 μg/NmL) reduced viability and increased cell death. O3SS treatment upregulated antioxidant genes, including Nrf2 and SOD1, and decreased reactive oxygen species in lipopolysaccharide (LPS)-stimulated microglia. Additionally, O3SS modulated microglial phenotype by reducing pro-inflammatory markers (iNOS, IL-1β) and increasing anti-inflammatory markers (Arg-1, IL-10). Immunofluorescence confirmed enhanced Arg-1 protein expression, indicating a shift toward an anti-inflammatory state. These results suggest that low-dose O3SS activates cellular antioxidant defenses and promotes an anti-inflammatory microglial phenotype, supporting its potential as a safe systemic O3 therapy. Further studies are warranted to confirm in vivo efficacy and optimize clinical protocols.