脑泰方对血红蛋白诱导的大鼠皮质神经元损伤的保护作用及机制  

Protective effect and its mechanism of Naotaifang on rat cortical neurons injury 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 Jinsong;Yu Jianbai;Liu Jian;Liao Jun;Luo Gang;Zhang Zhanwei;Huang Juan;Ge Jinwen(The First Hospital of Hunan University of Chinese Medicine,Hunan 410007,China;College of Integrated Traditional Chinese and Western Medicine,Hunan University of Chinese Medicine,Hunan 410208,China;Medical College of Hunan University of Chinese Medicine,Hunan 410208,China)

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

出　　处：《北京中医药大学学报》2019年第10期825-832,共8页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金资助项目(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(Hb)诱导的大鼠皮质神经元损伤的保护作用及其机制。方法原代培养新生SD大鼠皮质神经元,采用Hb诱导神经元模拟建立脑出血后神经元损伤细胞模型;实验设正常组、模型组、空白血清对照组、去铁胺组和10%脑泰方含药血清组。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;采用免疫荧光与高内涵细胞成像分析技术(HCA)检测各组神经元转铁蛋白(Tf)及其受体(TfR)、二价金属离子转运体(DMT-1)蛋白表达情况;采用酶联免疫吸附法检测各组上清液中脂质活性氧自由基(lipid-ROS)含量;采用real-time PCR法检测各组Tf、TfR和DMT-1 mRNA表达水平。结果与正常组比较,模型组神经元细胞活力明显降低(P<0.01),细胞内铁含量、细胞上清液中lipid-ROS水平显著升高(P<0.01),Tf、TfR及DMT1蛋白及mRNA表达水平明显上调(P<0.01)。与模型组比较,去铁胺组和10%脑泰方含药血清组细胞活力显著提高(P<0.01或P<0.05),细胞内铁含量、细胞上清液中lipid-ROS水平明显降低(P<0.01),Tf、TfR及DMT-1蛋白及其mRNA表达显著下调(P<0.01或P<0.05),去铁胺组和10%脑泰方含药血清组比较差异无统计学意义(P>0.05)。结论脑泰方可通过调节Hb诱导的大鼠皮质神经元细胞铁代谢,减轻神经元铁超载及lipid-ROS累积,从而提高神经元细胞活力;其作用与铁死亡抑制剂去铁胺一致,提示脑泰方可能通过抑制脑出血后神经元铁死亡而发挥神经保护作用。Objective To investigate the protective effect and its mechanism of Naotaifang(Brain Blood-activating Formula,NTF)on rat cortical neurons injury induced by hemoglobin(Hb)in vitro.MethodsPrimarycortical neuronsfrom newborn SD rats were cultured and induced by Hb(10 micmol/L)to simulate iron overload of neurons after ICH.The cultured neurons were randomly divided into the normal group,model group,blank serum group,10%NTF gmedicated serum roup and deferoxamine(DFX)group.Neuron viability was detected by CCK8 assay.The content of iron in neurons was detected by Calcein-AM staining.The protein expressions of transferring(Tf),transferrin receptor(TfR)and divalent metal ion transporter(DMT1)in neurons were detected by immunofluorescent staining and high content analysis(HCA).The content of lipid ROS was detected by using ELISA assay.The expressions of mRNA,Tf,TfR and DMT1 were detected with real-time PCR.Results Compared with the normal group,in the model group the neuron viability was markedly decreased(P<0.01),the iron and lipid ROS levels were significantly increased(P<0.01),and the expression of mRNA,Tf,TfR and DMT-1 were srgnificantly up-regulated(P<0.01).Compared with model group,in DFX and NTF groups,the neuron viability was srgnificantly increased(P<0.01 or P<0.05);the levels of iron and lipid ROS were significantly decreased(P<0.01);moreover,the expression levels of Tf,TfR,DMT-1 and mRNA were markedly down-regulated(P<0.01 or P<0.05).No significant difference was observed between the DFX group and 10%NTF group(P>0.05).Conclusion NTF might alleviate the iron overload and lipid ROS accumulation of Hb by regulating the iron metabolism in induced rat cortical neurons injury,so as to improve the activity of neurons.Its effect seems to be consistent with that of ferroptosis inhibitor DFX,suggesting that NTF could possibly exert neuroprotective effect by inhibiting ferroptosis of neurons after ICH.

关 键 词：脑泰方 脑出血 神经元 铁代谢 铁死亡 大鼠 

分 类 号：R285.5[医药卫生—中药学]