摘要
背景:铁死亡是脊髓缺血再灌注损伤的重要病理机制。研究证实,前列腺素E1可减轻慢性脑灌注不足诱导的海马体中脑微血管内皮细胞损伤,但它对脊髓缺血再灌注损伤后内皮细胞铁死亡的作用尚缺乏深入研究。目的:探讨前列腺素E1预处理能否通过抑制内皮细胞的铁死亡来减轻脊髓缺血再灌注损伤,并阐明可能的机制。方法:①细胞实验:将大鼠脊髓微血管内皮细胞分4组处理:对照组在常氧(体积分数20%O_(2))条件下加入完全培养基培养;模型组置于缺氧培养箱(体积分数95%N_(2)、5%CO_(2))中加入无葡萄糖无血清培养基3 h(氧-葡萄糖剥夺),随后在常氧(体积分数20%O_(2))条件下加入完全培养基培养12 h(复氧),建立氧-葡萄糖剥夺/复氧模型模拟脊髓缺血再灌注损伤;预处理组氧-葡萄糖剥夺3 h后继续培养2 h,添加前列腺素E1处理2 h后复氧培养12 h;抑制剂组氧-葡萄糖剥夺3 h后添加核因子红细胞2相关因子2抑制剂ML385处理2 h,再添加前列腺素E1处理2 h,复氧培养12 h。处理结束后,检测细胞内丙二醛、谷胱甘肽和Fe2+浓度,CCK-8法检测细胞活力,免疫荧光染色与Western blot检测酰基辅酶A合成酶长链家族成员4、谷胱甘肽过氧化物酶4表达,流式细胞术检测细胞内活性氧水平,Western blot检测核因子红细胞2相关因子2、血红素加氧酶1蛋白表达。②动物实验:将45只大鼠随机分为3组处理:假手术组(n=15)仅开腹,暴露腹主动脉但不阻断;模型组(n=15)夹闭腹主动脉30 min后经尾静脉注射生理盐水,随后恢复动脉血流(建立脊髓缺血再灌注损伤模型);预处理组(n=15)夹闭腹主动脉30 min后经尾静脉注射前列腺素E1,随后恢复动脉血流。恢复动脉血流24 h后,通过BBB评分、斜板实验和尼氏染色评估大鼠运动功能和神经元损伤情况,脊髓含水量检测、闭锁小带蛋白免疫荧光染色和CD34免疫组化染色评估血脊髓屏障完整性和微血管密度,免疫荧光染色、普鲁士蓝染色、Western blot和生化检测评估脊髓组织铁死亡情况。结果与结论:①细胞实验:氧-葡萄糖剥夺/复氧造模可降低大鼠脊髓微血管内皮细胞活力、诱导细胞铁死亡以及下调核因子红细胞2相关因子2、血红素加氧酶1蛋白表达,前列腺素E1预处理可抑制氧-葡萄糖剥夺/复氧造模对大鼠脊髓微血管内皮细胞的上述影响;ML385部分逆转了前列腺素E1预处理的作用。②动物实验:前列腺素E1预处理可减轻脊髓缺血再灌注损伤造成的大鼠运动功能缺陷与神经元损伤、血脊髓屏障损伤,改善微血管密度,抑制脊髓组织铁死亡。③结果表明,前列腺素E1预处理通过激活核因子红细胞2相关因子2/血红素加氧酶1信号通路抑制内皮细胞的铁死亡,对脊髓缺血再灌注损伤发挥保护作用。
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%O_(2))with complete medium.Model group was cultured in a hypoxic incubator(95%N_(2) and 5%CO_(2))with glucose-free and serum-free medium for 3 hours(oxygen-glucose deprivation),and then in normoxia(20%O_(2))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.
作者
黄玉珊
王荣荣
李湘淼
白金柱
Huang Yushan;Wang Rongrong;Li Xiangmiao;Bai Jinzhu(School of Rehabilitation Medicine,Capital Medical University,Beijing 100068,China;Department of Spine and Spinal Cord Surgery,Beijing Boai Hospital,China Rehabilitation Research Center,Beijing 100068,China;College of Orthopedics,Capital Medical University,Beijing 100069,China)
出处
《中国组织工程研究》
北大核心
2026年第22期5716-5727,共12页
Chinese Journal of Tissue Engineering Research
基金
中国康复研究中心重点项目(2022ZX-05,2018ZX-08),项目负责人:白金柱。
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
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
