脑泰方对血红蛋白诱导的大鼠皮质神经元铁超载及过氧化损伤的多靶点干预作用  

Effects of Naotai Formula on Iron Overload and Peroxidation Injury in Cortical Neurons of Rats Induced by Hemoglobin

作　　者：曾劲松[1,2] 喻坚柏 刘检[1] 廖君[3] 罗刚 张占伟[1] 黄娟[3] 葛金文 ZENG Jin-song;YU Jian-bai;LIU Jian;LIAO Jun;LUO Gang;ZHANG Zhan-wei;HUANG Juan;GE Jin-wen(Department of Neurosurgery,First Hospital of Hunan University of Chinese Medicine,Changsha,410007;College of Integrated Traditional Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha,410007;Medical College,Hunan University of Chinese Medicine,Changsha,410007)

机构地区：[1]湖南中医药大学第一附属医院神经外科,长沙410007 [2]湖南中医药大学中西医结合学院,长沙410208 [3]湖南中医药大学医学院,长沙410208

出　　处：《中国中西医结合杂志》2020年第6期708-714,共7页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金项目(No.81774174,No.81774033);湖南省中医药科研项目(No.201969);湖南中医药大学中西医结合一流学科开放基金项目(No.2018ZXYJH15)。

摘　　要：目的探讨脑泰方对体外血红蛋白(HGB)诱导的大鼠皮质神经元铁超载及过氧化损伤的多靶点干预作用及其机制。方法原代培养新生SD大鼠皮质神经元,采用HGB诱导神经元模拟建立脑出血后神经元铁超载及氧化应激损伤细胞模型;实验设正常组、模型组、空白血清对照组、铁离子螯合剂甲磺酸去铁胺(DFX)组、抗氧化剂N-乙酰半胱氨酸(NAC)组和脑泰方含药血清(NTF)组。采用CCK8法检测各组细胞活力;采用Calcein-AM染色法检测各组细胞内铁含量;采用ELISA法检测各组谷胱甘肽(GSH)含量、谷胱甘肽过氧化物酶4(GPX-4)活性及各组上清液中脂质活性氧(lipid ROS)含量;采用高内涵细胞成像分析技术(HCA)检测各组神经元转铁蛋白受体(TfR)、铁调节蛋白2(IRP-2)、膜铁转运蛋白1(Fpn-1)表达。结果与正常组比较,模型组及空白血清对照组神经元细胞活力降低(P<0.01),细胞内铁含量、细胞上清液中lipid ROS含量、TfR蛋白及Fpn-1蛋白表达升高(P<0.01,P<0.05),IRP-2、GSH含量、GPX-4活性降低(P<0.01,P<0.05)。与模型组比较,DFX组、NAC组和NTF组细胞活力、GSH含量、GPX-4活性升高(P<0.01,P<0.05),细胞内铁含量、细胞上清液lipid ROS含量降低(P<0.05);DFX组及NTF组细胞TfR及IRP-2蛋白表达降低(P<0.01,P<0.05),Fpn-1蛋白表达升高(P<0.01,P<0.05)。结论脑泰方可通过减轻HGB诱导的大鼠皮质神经元细胞铁超载及lipid ROS累积,从而提高神经元细胞活力;其作用机制与铁死亡的生物学过程相吻合。脑泰方可能通过对神经细胞铁代谢及抗氧化信号分子的多靶点干预作用,抑制脑出血后神经细胞铁死亡而发挥神经保护作用。Objective To explore the multi-target intervention effect and mechanism of Naotai Formula(NTF)on hemoglobin(HGB)-induced iron overload and peroxidative damage in rat cortical neuronsin vitro.Methods Cortical neurons of newborn SD rats were cultured,and HGB-induced neuron simulation was used to es-tablish a neuronal iron overload and oxidative stress injury cell model after cerebral hemorrhage.The rats were divided into normal group,model group,blank serum control group,Deferoxamine(DFX)group,N-acetylcysteine(NAC)group and NTF serum containing group.Cell viability was measured by CCK8 method,intracellular iron content was measured by Calcein-AM staining,glutathione(GSH),glutathione peroxidase 4(GPX-4)activity and lipid reactive oxygen species(lipid ROS)content in supernatants were measured by ELISA.The expression of neuronal transferrin receptor(TfR),iron regulatory protein 2(IRP-2)and ferroportin 1(Fpn-1)were detected by immunofluorescence and high connotation cell imaging analysis(HCA).Results Compared with normal group,the viability of neurons decreased significantly in the model group and blank serum control group(P<0.01),the level of intracellular iron content,and the expression of lipid ROS,TfR protein and Fpn-1 protein increased(P<0.01,P<0.05),IRP-2,GSH content and GPX-4 activity decreased(P<0.01,P<0.05).Compared with model group,the cell viability,GSH content,and GPX-4 activity were significantly increased in DFX,NAC and NTF groups(P<0.01,P<0.05),the intracellular iron content and lipid ROS in cell supernatant were decreased(P<0.05),the level of TfR and IRP-2 were decreased in DFX and NTF groups(P<0.01,P<0.05),Fpn-1 levels increased(P<0.01,P<0.05).Conclusions NTF can increase the activity of neurons by reducing iron overload and lipid ROS accumulation in cortical neurons of rats induced by HGB,its mechanism is consistent with the biological process of ferroptosis.NTF may play a neuroprotective role by interfering with iron metabolism and antioxidant signaling molecules and inhibiting ferroptosis of nerve c

关 键 词：脑泰方 脑出血 神经元 抗氧化 铁代谢 铁死亡 

分 类 号：R285.5[医药卫生—中药学]