Ozone-Mediated Cerebral Protection: Unraveling the Mechanism through Ferroptosis and the NRF2/SLC7A11/GPX4 Signaling Pathway

The pathogenesis of brain ischemic/reperfusion (I/R) insult is characterized by neuronal loss due to excessive oxidative stress responses. Ferroptosis, a form of oxidative cell death, can be triggered when the balance between antioxidants and pro-oxidants in cells is disrupted. Ozone, a natural bioactive molecule with antioxidant/anti-apoptotic and pro-autophagic properties, has been shown to enhance the antioxidant system's capacity and ameliorate oxidative stress. However, its role in neuronal ferroptosis remains unclear. Therefore, we investigated the functions and possible mechanisms of ozone in cerebral I/R-induced ferroptotic neuronal death. A cerebral ischemia-reperfusion injury model was induced in Sprague-Dawley (SD) rats pre-treated with ozone. Intraperitoneal administration of the NRF2 inhibitor ML385, the SLC7A11 inhibitor Erastin, and the GPX4 inhibitor RSL3 was performed one hour prior to model establishment. Our results showed that ozone preconditioning mitigated neuronal damage caused by cerebral I/R, reduced the severity of neurological deficits, lowered cerebral infarct volume in middle cerebral artery occlusion (MCAO) rats, and decreased the volume of cerebral infarcts. Transmission electron microscopy, immunofluorescence, and Western blotting indicated ferroptosis following MCAO-induced brain damage. MCAO resulted in morphological damage to neuronal mitochondria, increased lipid peroxidation accumulation, and elevated malondialdehyde (MDA) production. Furthermore, MCAO decreased levels of FTH1 and GPX4 (negative regulators of ferroptosis) and increased ACSL4 levels (a positive regulator of ferroptosis). Ozone preconditioning demonstrated a neuroprotective effect by increasing NRF2 nuclear translocation and the expression of SLC7A11 and GPX4. Treatment with ML385, Erastin, and RSL3 significantly reversed ozone preconditioning's protective effect on neuronal ferroptosis. Our findings demonstrated that ozone treatment attenuates ferroptosis in a cerebral ischemia/reperfusion injury rat model via the NRF2/SLC7A11/GPX4 pathway, providing a theoretical basis for ozone's potential use as a therapy to prevent ischemic stroke.