[Effect of electroacupuncture pretreatment on ferroptosis in neurons of rats with cerebral ischemia-reperfusion injury].

To observe the effect of electroacupuncture（EA）preconditioning on ferroptosis in rats with cerebral ischemia-reperfusion injury （CIRI）, so as to explore the neuroprotective mechanism of EA preconditioning.Male SD rats were randomly divided into sham operation, model, EA, inhibitor and inducer groups with 20 rats in each group. The CIRI model was established by modified Zea Longa occlusion of the middle cerebral artery. Before modeling, EA treatment （2 Hz/15 Hz, 1-2 mA） was applied to "Baihui"（GV20）, "Fengfu"（GV16） and "Dazhui"（GV14） for rats of the EA group, 20 min a day for 7 consecutive days. Rats of the inhibitor group were intraperitoneally injected with ferristatin-1（25 mg/kg）at a slow and uniform rate. Rats of the inducer group were intraperitoneally injected with Erastin（100 mg/kg） after 7 days of EA preconditioning, once every 2 h for a total of 4 times. The CIRI models were prepared 2 d later after the above interventions finished by thread-occlusion. The degree of neurological impairment was evaluated by modified Zea Longa score. The percentage of infarct size was calculated by TTC staining. The ultrastructure of neurons in hippocampus was observed by transmission electron microscope. The contents of ferrous ion （Fe2+）, malondialdehyde （MDA） and glutathione （GSH） in cerebral tissue and reactive oxygen species （ROS） in serum were determined by biochemical method. The changes of mitochondrial membrane potential in rats brain tissues were detected by flow cytometry. The mRNA and protein expression levels of glutathione peroxidase 4 （GPX4）, acyl-CoA synthetase long-chain family member 4 （ACSL4）, transferrin receptor （TFRC）, 15-lipoxygenase （15-LOX） and cyclooxygenase-2 （COX-2） in the ischemic hippocampal region of CIRI rats were detected by real-time quantitative PCR and Western blot, respectively.Compared with the sham operation group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression levels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01）, and mitochondria in brain tissue were significantly damaged （P<0.01） in the model group. Compared with the model group, the above indexes were all reversed （P<0.05, P<0.01） in the EA group and inhibitor group. Compared with the EA group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression le-vels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.05, P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01, P<0.05）, and mitochondria in brain tissue were significantly damaged （P<0.05） in the inducer group.EA preconditioning has neuroprotective effect on CIRI rats, which may be related to inhibiting ACSL4/TFRC/15-LOX/COX-2 expression and increasing GSH/GPX4 expression.目的：观察电针预处理对脑缺血再灌注损伤（CIRI）大鼠神经元铁死亡的影响，探讨电针预处理对神经元的保护作用机制。方法：将雄性SD大鼠随机分为假手术组、模型组、电针组、抑制剂组和诱导剂组，每组20只。采用改良Zea Longa线栓法制备CIRI大鼠模型。造模前，电针组予“百会”“风府”“大椎”电针刺激，每天20 min，连续7 d；抑制剂组腹腔注射铁抑素-1；诱导剂组于电针治疗7 d后腹腔注射埃拉斯汀。各组干预结束2 d后造模。采用改良Zea Longa法进行神经功能缺损评分；TTC染色法评估脑梗死面积并计算梗死面积百分比；透射电镜观察海马组织神经细胞超微结构；生化法检测缺血区脑组织亚铁离子（Fe2+）、丙二醛（MDA）、谷胱甘肽（GSH）及血清活性氧（ROS）的含量；流式细胞仪检测大鼠脑组织线粒体膜电位变化；实时荧光定量PCR法及Western blot法检测缺血区海马组织谷胱甘肽过氧化物酶4（GPX4）、酯酰辅酶A合成酶长链家族成员4（ACSL4）、转铁蛋白受体（TFRC）、15-脂氧合酶（15-LOX）、环氧化酶2（COX-2）的mRNA和蛋白表达水平。结果：与假手术组比较，模型组大鼠神经功能缺损评分、脑梗死面积百分比、脑组织MDA和Fe2+含量、血清ROS含量、海马组织ACSL4、TFRC、15-LOX、COX-2蛋白及mRNA表达水平升高（P<0.01），脑组织GSH含量、海马组织GPX4蛋白和mRNA表达水平降低（P<0.01），脑组织中线粒体显著损伤（P<0.01）；与模型组比较，电针组、抑制剂组大鼠神经功能缺损评分、脑梗死面积百分比、脑组织MDA和Fe2+含量、血清ROS含量、海马组织ACSL4、TFRC、15-LOX、COX-2蛋白及mRNA表达水平降低（P<0.05，P<0.01），脑组织GSH含量、海马组织GPX4蛋白和mRNA表达水平升高（P<0.01），脑组织线粒体损伤程度减轻（P<0.05，P<0.01）；与电针组比较，诱导剂组大鼠神经功能缺损评分、脑梗死面积百分比、脑组织MDA和Fe2+含量、血清ROS含量、海马组织ACSL4、TFRC、15-LOX、COX-2蛋白及mRNA表达水平升高（P<0.05，P<0.01），脑组织GSH含量、海马组织GPX4蛋白和mRNA表达水平显著降低（P<0.01，P<0.05），脑组织线粒体显著损伤（P<0.05）。结论：电针预处理对CIRI大鼠具有神经保护作用，这可能与升高GSH/GPX4表达，抑制ACSL4/TFRC/15-LOX/COX-2表达从而抑制神经元铁死亡有关。.