关键词: 前列腺素E1, 预处理, 脊髓缺血再灌注损伤, 铁死亡, 内皮细胞, 血脊髓屏障, 神经功能, Nrf2/HO-1信号通路

Abstract: BACKGROUND: Ferroptosis is an important pathological mechanism in spinal cord ischemia-reperfusion injury. Although studies have confirmed that prostaglandin E1 attenuates cerebral microvascular endothelial cell injury in the hippocampus induced by chronic cerebral hypoperfusion, its effect on ferroptosis of endothelial cells after spinal cord ischemia-reperfusion injury remains poorly studied.
OBJECTIVE: To investigate whether prostaglandin E1 pretreatment attenuates spinal cord ischemia-reperfusion injury by inhibiting ferroptosis in endothelial cells and to elucidate possible mechanisms.
METHODS: (1) Cell experiment: Rat spinal cord microvascular endothelial cells were divided into four groups. Control group was cultured in normoxia (20% O2) with complete medium. Model group was cultured in a hypoxic incubator (95% N2 and 5% CO2) with glucose-free and serum-free medium for 3 hours (oxygen-glucose deprivation), and then in normoxia (20% O2) with complete medium for 12 hours (reoxygenation), to establish an oxygen-glucose deprivation/reoxygenation model to simulate spinal cord ischemia-reperfusion injury. Pretreatment group was subjected to oxygen-glucose deprivation for 3 hours and incubation for 2 hours, followed by treatment with prostaglandin E1 for 2 hours and reoxygenation for 12 hours. Inhibitor group was subjected to oxygen-glucose deprivation for 3 hours, followed by addition of nuclear factor erythroid 2-related factor 2 inhibitor ML385 for 2 hours, then incubated with prostaglandin E1 for 2 hours, and reoxygenated for 12 hours. At the end of treatment, intracellular malondialdehyde, glutathione and Fe2+ concentration were detected, cell viability was measured by cell counting kit-8 assay, immunofluorescence staining with western blot assay were detected the expressions of acyl-coenzyme A synthase long-chain family member 4, and glutathione peroxidase 4, the intracellular reactive oxygen species level was detected by flow cytometry, and Western blot was detected the protein expressions of nuclear factor erythroid 2-related factor 2, and heme oxygenase 1. 
(2)Animal experiment: 45 rats were randomly divided into three groups. Sham group (n = 15) only opened the abdomen to expose the abdominal aorta but did not block it. Model group (n = 15) clamped the abdominal aorta for 30 minutes and then injected normal saline via the tail vein, followed by the restoration of arterial blood flow to establish a model of spinal cord ischemia-reperfusion injury. Pretreatment group (n = 15) clamped the abdominal aorta for 30 minutes and then injected with prostaglandin E1 via the tail veinord, followed by restoration of arterial blood flow. Twenty-four hours after restoration of arterial blood flow, motor function and neuronal damage in rats were assessed by Basso-Beattie-Bresnahan score, inclined plane test and Nissl staining. The blood-spinal cord barrier integrity and microvessel density were assessed by spinal cord water content measurement, zonula occludens-1 (ZO-1) immunofluorescence staining and CD34 immunohistochemical staining. The ferroptosis in spinal cord tissue was assessed by immunofluorescence staining, Prussian blue staining, Western blot and biochemical assays. 
RESULTS AND CONCLUSION: (1) Cell experiment: Oxygen-glucose deprivation/reoxygenation decreased the viability of rat spinal cord microvascular endothelial cells, induced cellular ferroptosis, and down-regulated the protein expressions of nuclear factor erythroid 2-related factor 2, and heme oxygenase 1, and the above effects of oxygen-glucose deprivation/reoxygenation on rat spinal cord microvascular endothelial cells were suppressed by pretreatment with prostaglandin E1. The effects of prostaglandin E1 pretreatment were partially reversed by ML385. (2) Animal experiment: Prostaglandin E1 pretreatment attenuated motor function deficits, neuronal damage, and blood-spinal cord barrier damage, improved microvessel density, and inhibited ferroptosis in spinal cord tissues in rats caused by spinal cord ischemia-reperfusion injury. Overall, prostaglandin E1 pretreatment protects against spinal cord ischemia-reperfusion injury by inhibiting ferroptosis in endothelial cells through activation of the nuclear factor erythroid 2-related factor 2/heme oxygenase 1 signaling pathway.

Key words: prostaglandin E1, pretreatment, spinal cord ischemia-reperfusion injury, ferroptosis, endothelial cells, blood-spinal cord barrier, neurological function, nuclear factor erythroid 2-related factor 2/heme oxygenase 1 signaling pathway