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.

中文翻译：

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臭氧介导的脑保护：通过铁死亡和 NRF2/SLC7A11/GPX4 信号通路揭示机制

脑缺血/再灌注（I/R）损伤的发病机制的特点是过度氧化应激反应导致神经元损失。当细胞中抗氧化剂和促氧化剂之间的平衡被破坏时，就会触发铁死亡，这是氧化性细胞死亡的一种形式。臭氧是一种天然生物活性分子，具有抗氧化/抗凋亡和促自噬特性，已被证明可以增强抗氧化系统的能力并改善氧化应激。然而，其在神经元铁死亡中的作用仍不清楚。因此，我们研究了臭氧在脑缺血再灌注引起的铁死亡神经元死亡中的功能和可能机制。在用臭氧预处理的SD大鼠中诱导脑缺血再灌注损伤模型。模型建立前1小时腹腔内给予NRF2抑制剂ML385、SLC7A11抑制剂Erastin和GPX4抑制剂RSL3。我们的结果表明，臭氧预处理减轻了脑缺血再灌注引起的神经元损伤，降低了神经功能缺损的严重程度，降低了大脑中动脉闭塞（MCAO）大鼠的脑梗塞体积，并减少了脑梗塞体积。透射电子显微镜、免疫荧光和蛋白质印迹表明 MCAO 引起的脑损伤后出现铁死亡。 MCAO 导致神经元线粒体形态损伤、脂质过氧化积累增加和丙二醛 (MDA) 产量增加。此外，MCAO 降低了 FTH1 和 GPX4（铁死亡的负调节因子）的水平，并增加了 ACSL4（铁死亡的正调节因子）的水平。臭氧预处理通过增加 NRF2 核转位以及 SLC7A11 和 GPX4 的表达证明了神经保护作用。 ML385、Erastin 和 RSL3 治疗显着逆转了臭氧预处理对神经元铁死亡的保护作用。我们的研究结果表明，臭氧治疗通过NRF2/SLC7A11/GPX4途径减轻脑缺血/再灌注损伤大鼠模型中的铁死亡，为臭氧作为预防缺血性中风的潜在疗法提供了理论基础。