The Role of Phosphatidylethanolamine Binding Protein 1/15-Lipoxygenase 2/Glutathione Peroxidase 4 Signaling Pathway in Cerebral Ischemia Reperfusion-Induced Ferroptosis; In Vitro and in Vivo Study

Abstract

Objective: To investigate the role of phosphatidylethanolamine binding protein 1/15-Lipoxygenase 2/Glutathione Peroxidase 4 (PEBP1/15-lox2/GPX4) signaling pathway in cerebral ischemia reperfusion (I/R)-induced ferroptosis. Methods: The expression of PEBP1, 15-lox2, GPX4 and SLC7A11 in the peripheral serum samples of stroke patients and healthy controls was detected with quantitative polymerase chain reaction (qPCR). HT22 was selected to establish the hypoxia reoxygenation model. Enzyme linked immunosorbent assay (ELISA) was applied to detect the levels of superoxide dismutase (SOD) and malondialdehyde (MDA), transmission electron microscope to observe the microstructure of mitochondria, and western blotting to detect the expression levels of SLC7A11, 15-lox2, GPX4 and PEBP1, co-immunoprecipitation (CO-IP) to verify the interaction of PEBP1 with 15-lox2. Additionally, the function of PEBP1/15-lox2/GPX4 signaling pathway was further investigated in cerebral I/R-induced injury. Results: The expression of PEBP1 and 15-lox2 was significantly higher, while the expression of GPX4 and SLC7A11 was significantly lower in stroke patients, than that in healthy controls (p < 0.05). In HT22 cells, hypoxia reoxygenation significantly promoted MDA level and significantly reduced SOD level, compared with control. Additionally, hypoxia reoxygenation significantly increased PEBP1, 15-lox2 expression and p-PEBP1 level, and significantly reduced GPX4 and SLC7A11 expression (p < 0.05). These changes were significantly relieved by PEBP1 knockdown and significantly aggravated by PEBP1 overexpression. Morphological results also showed that PEBP1 knockdown also prevented hypoxia reoxygenation-induced cell injury. Co-Immunoprecipitation (CO-IP) also indicated a direct interaction between PEBP1 and 15-lox2. In vivo data showed that cerebral I/R significantly increased the infarct size, MDA level, PEBP1 and 15-lox2 expression, p-PEBP level, and significantly reduced SOD, GPX4 and SLC7A1 expression, compared with sham group. PEBP1 knockdown significantly attenuated those changes (p < 0.05). Morphological results further indicated that PEBP1 knockdown prevented cerebral I/R-induced injury. Conclusions: PEBP1/15-lox2/GPX4 signaling pathway participates in cerebral ischemia reperfusion-induced ferroptosis.