Keap1/Nrf2/HO-1/GPX4信号通路阻断癫痫大鼠海马神经元铁死亡机制及草果知母汤的干预效应  

The Mechanism of Keap1/Nrf2/HO-1/GPX4 Signal Pathway Blocking Iron Death in Epileptic Rats Hippocampal Neurons and the Intervention Effect of Caoguo Anemarrhena Decoction

作　　者：梁韡斌 张高炼[1] 郭建辉 曾敬 李敏[1] 陈锐[1] 张晓宁[1] 刘姗姗[1] LIANG Weibin;ZHANG Gaolian;GUO Jianhui;ZENG Jing;LI Min;CHEN Rui;ZHANG Xiaoning;LIU Shanshan(First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine,Nanning 530012,Guangxi,China)

机构地区：[1]广西中医药大学第一附属医院,南宁530012

出　　处：《中西医结合心脑血管病杂志》2022年第18期3323-3329,共7页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：广西自然科学基金项目(No.2019GXNSFAA245088)。

摘　　要：目的 探讨Kelch-样ECH相关蛋白1(Keap1)/核因子E2相关因子2(Nrf2)/血红素加氧酶-1(HO-1)/谷胱甘肽过氧化物酶4(GPX4)信号通路阻断癫痫海马神经元铁死亡机制及草果知母汤的干预效应。方法 将60只大鼠随机分成正常对照组、模型组、草果知母汤低、中、高剂量组和西药对照组,每组10只。采用腹腔注射戊四氮(PTZ)法建立癫痫大鼠模型,草果知母汤低、中、高剂量组分别给予草果知母汤1.25 g/kg、2.50 g/kg、5.00 g/kg,西药对照组腹腔注射丙戊酸钠(200 mg/kg),连续给药5周,对大鼠进行Racine评分,使用酶联免疫吸附法(ELISA)试剂盒测定海马组织谷胱甘肽(GSH)、活性氧(ROS)及铁含量,苏木精-伊红(HE)及尼氏(Nissl)染色法观察海马组织病理学变化并计算海马神经元细胞数,免疫组化检测海马组织CA1区GPX4、Nrf2蛋白表达积分光密度(IOD),实时荧光定量聚合酶链式反应(RTq-PCR)及免疫印迹法(Western Blot)检测海马组织Keap1、GPX4、Nrf2及HO-1 mRNA及蛋白表达水平。结果 与正常对照组比较,模型组大鼠Racine评分、海马组织铁含量及ROS水平、海马组织Keap1 mRNA及蛋白水平均升高(P<0.05),海马组织GSH水平、海马神经元细胞数、海马组织GPX4、Nrf2及HO-1 mRNA及蛋白表达水平、海马CA1区GPX4、Nrf2蛋白免疫组化积分光密度降低(P<0.05)。与模型组比较,草果知母汤低、中、高各剂量及西药对照组大鼠Racine评分、海马组织铁含量及ROS水平、海马组织Keap1 mRNA及蛋白水平降低(P<0.05),海马组织GSH水平、海马神经元细胞数、海马组织GPX4、Nrf2及HO-1 mRNA水平及蛋白表达水平、海马CA1区GPX4、Nrf2蛋白表达积分光密度升高(P<0.05)。结论 草果知母汤能够修复癫痫大鼠海马组织神经元氧化损伤,其机制可能与调节Keap1/Nrf2/HO-1/GPX4通路进而阻断海马神经元铁死亡有关。Objective To explore iron death of epileptic rats hippocampal neurons and the intervention effect of Caoguo Anemarrhena Decoction.Methods Sixty rats were randomly divided into normal control group, model group, low, middle, and high dose groups of Caoguo Anemarrhena Decoction, and western medicine control group, with 10 rats in each group.The rat model of epilepsy was established by intraperitoneal injection of pentylenetetrazol(PTZ).The rats in the low, middle, and high dose groups were given Caoguo Anemarrhena Decoction according to 1.25 g/kg, 2.50 g/kg, and 5.00 g/kg, respectively, and the rats in western medicine control group were injected intraperitoneally with sodium valproate(200 mg/kg) for 5 weeks.The rats were scored by Racine.The contents of glutathione(GSH),reactive oxygen species(ROS),and iron in hippocampus were measured by enzyme-linked immunosorbent assay(ELISA) kit.The histopathological changes of hippocampus were observed by hematoxylin-eosin(HE) and Nissl staining, and the number of hippocampal neurons was calculated.The integrated optical density(IOD) of GPX4 and Nrf2 protein in hippocampal CA1 region was detected by immunohistochemistry.The expression level and protein and mRNA of Kelch-like epichlorohydrin-associated protein(Keap1),glutathione peroxidase 4(GPX4),nuclear factor E2 related factor 2(Nrf2),and heme oxygenase-1(HO-1) in hippocampal tissue were detected by real-time quantitative polymerase chain reaction(RTq-PCR) and Western Blot.Results Compared with normal control group, the Racine score, iron content, and ROS level in the hippocampus, the Keap mRNA and protein level in the hippocampus were increased significantly(P<0.05),the GSH level in the hippocampus, the number of hippocampal neurons, the mRNA and protein expression levels of GPX4,Nrf2,and HO-1 mRNA in the hippocampus, the immunohistochemical integral optical density of GPX4 and Nrf2 proteins in the hippocampal CA1 region were decreased in model group(P<0.05).Compared with model group, Racine score, iron content, and ROS le

关 键 词：癫痫 草果知母汤 Kelch-样ECH相关蛋白1/核因子E2相关因子2/血红素加氧酶-1/谷胱甘肽过氧化物酶4通路 铁死亡 实验研究 

分 类 号：R742.1[医药卫生—神经病学与精神病学]