基于铁死亡探讨温阳复元方对脑缺血再灌注损伤大鼠神经损伤的保护机制  

Protective Mechanism of Wenyang Fuyuan Prescription on Nerve Injury in Rats with Cerebral IschemiaReperfusion Injury Based on Ferroptosis

作　　者：向军军[1,2] 李丽琴 李建铮 莫雪妮[2] 陈炜[1] 胡跃强[1] XIANG Junjun;LI Liqin;LI Jianzheng;MO Xueni;CHEN Wei;HU Yueqiang(The First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine,Nanning 530023 Guangxi,China;Guangxi University of Traditional Chinese Medicine,Nanning 530000 Guangxi,China)

机构地区：[1]广西中医药大学第一附属医院,广西南宁530023 [2]广西中医药大学,广西南宁530000

出　　处：《中药新药与临床药理》2023年第12期1649-1657,共9页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：广西中医药大学2021年研究生教育创新计划项目(YCBXJ2021008);国家自然科学基金项目(81973768)。

摘　　要：目的基于铁死亡探讨中药温阳复元方改善脑缺血再灌注损伤(Cerebral ischemia-reperfusion injury,CIRI)大鼠神经损伤的可能机制。方法SD大鼠72只,随机分为假手术组、模型组、温阳复元方组、铁死亡诱导剂组、温阳复元方+铁死亡诱导剂组、铁死亡抑制剂组,每组12只。假手术组手术步骤同模型组,但线栓只插入颈内动脉深度9 mm,不堵塞大脑中动脉,其余各组采用线栓法构建大脑中动脉闭塞/再灌注(middle cerebral artery occlusion/reperfusion,MCAO/R)模型。在造模前24 h开始,按照分组给予铁死亡诱导剂(100 mg·kg^(-1))、铁死亡抑制剂(5 mg·kg^(-1))腹腔注射;中药温阳复元方(18.0 g·kg^(-1))灌胃于麻醉清醒后2 h开始。各干预措施每天1次,连续7 d。分别于MCAO/R术后第1、3、7天采用Longa评分标准进行神经功能缺损评分。疗程结束后取材,采用苏木素-伊红染色法(HE)观察各组大鼠神经元形态学变化,透射电子显微镜观察各组大鼠神经元超微结构变化,生化试剂盒检测脑组织亚铁离子(Fe^(2+))和还原型谷胱甘肽(GSH)含量的变化,实时荧光定量聚合酶链式反应(RT-qPCR)、Western Blot法检测转运铁蛋白受体1(TFR1)、铁调节蛋白1(IRP1)、膜铁转运蛋白(FPN)mRNA及蛋白的表达变化。结果(1)与假手术组比较,模型组大鼠在各时间点神经功能缺损评分均升高(P<0.01);HE染色显示神经元稀疏杂乱、细胞核固缩、边缘出现空泡,电镜下可见神经元线粒体数目减少、线粒体膜密度增加或破裂溶解、线粒体嵴消失;Fe2+含量与TFR1 mRNA及蛋白表达量升高(P<0.01),GSH含量与IRP1、FPN mRNA及蛋白表达量明显下降(P<0.05,P<0.01)。(2)与模型组比较,温阳复元方组大鼠在各时间点神经功能缺损评分降低(P<0.05);神经元数目增多且排列相对整齐、细胞核形态完整清晰,神经元线粒体结构相对完整、线粒体膜相对完整、线粒体嵴清晰;Fe^(2+)含量与TFR1 mRNA及蛋�Objective To investigate the protective mechanism of Wenyang Fuyuan Prescription on nerve injury by improving brain iron metabolism in rats with cerebral ischemia-reperfusion injury(CIRI)based on ferroptosis.Methods A total of 72 SD rats were randomly divided into sham-operation group,CIRI model group,Wenyang Fuyuan Prescription group(18.0 g·kg^(-1),gavage),ferroptosis inducer group(100 mg·kg^(-1),intraperitoneal injection),Wenyang Fuyuan Prescription(18.0 g·kg^(-1),gavage)+ferroptosis inducer group(intraperitoneal injection)and ferroptosis inhibitor group(5 mg·kg^(-1),intraperitoneal injection),12 rats in each group.All the procedures adopted in the sham group were the same as those in the model group.But nylon thread was inserted into the internal carotid artery at a depth of 9 mm and un-plugged middle cerebral artery.The rest of the groups were used to construct middle cerebral artery occlusion/reperfusion(MCAO/R)model by thread embolism method.Ferroptosis inducer(100 mg·kg^(-1))and ferroptosis inhibitor(5 mg·kg^(-1))were administered intraperitoneally to rats according to the grouping 24 hours before modeling.Wenyang Fuyuan Prescription(18.0 g·kg^(-1))was administered by gavage 2 hours after anesthesia and awakening.All intervention were given once daily for7 consecutive days.The Longa scoring standard was used to evaluate the neurological deficit on 1,3,and 7 days after MCAO/R surgery,respectively.At the end of the treatment period,brain tissues were taken to observe the morphological changes of rat neurons in each group by hematoxylin eosin staining(HE).The ultrastructural changes of neuron mitochondria in each group were observed by transmission electron microscope.The biochemical kit was used to detect the content of iron ions(Fe^(2+))and reduced glutathione(GSH)in brain tissue.The protein and mRNA expressions of transferrin receptor 1(TFR1),iron regulatory protein 1(IRP1)and ferroportin(FPN)were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western Blot.Results(1)Compare

关 键 词：温阳复元方 脑缺血再灌注损伤 神经损伤 脑铁代谢 铁死亡 铁调节蛋白1(IRP1) 大鼠 

分 类 号：R285.5[医药卫生—中药学]