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 <i>Nrf2</i> and <i>SOD1</i>, and decreased reactive oxygen species in lipopolysaccharide (LPS)-stimulated microglia. Additionally, O₃SS modulated microglial phenotype by reducing pro-inflammatory markers (<i>iNOS</i>, <i>IL-1β</i>) and increasing anti-inflammatory markers (<i>Arg-1</i>, <i>IL-10</i>). 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.