脑出血后血红素通过白介素-1受体诱导神经元发生程序性坏死  

Hemin induces necroptosis of neurons through interleukin-1 receptor after intracerebral hemorrhage

作　　者：吴晓凤[1] 初翔 谭燕[1] 李磊[1] Wu Xiaofeng;Chu Xiang;Tan Yan;Li Lei(First Laboratory, Research Institute of Surgery, Daping Hospital, Army Medical University, State Key Lab of Trauma, Burns and Combined Injury, Chongqing 400042, China)

机构地区：[1]陆军军医大学大坪医院野战外科研究所第一研究室,创伤、烧伤与复合伤国家重点实验室,重庆400042

出　　处：《中华神经医学杂志》2019年第8期757-766,共10页Chinese Journal of Neuromedicine

基　　金：国家重点基础研究发展计划(973计划)(2014CB541605).

摘　　要：目的探讨脑出血后游离血红素对神经元死亡的影响及其可能的分子机制。方法(1)实验一:将24只C57BL/6小鼠按随机数字表法分为假手术组、生理盐水组、5 mmol/L血红素组、10 mmol/L血红素组,每组6只。后2组分别注射20 μL 5 mmol/L、10 mmol/L血红素溶液至纹状体制备成脑出血模型,生理盐水组注射20 μL生理盐水。12 h后采用Western blotting检测海马组织中血红素氧合酶-1(HO-1)及受体间相互作用蛋白(RIP)1、RIP3、混合谱系激酶域样蛋白(MLKL)的蛋白表达。将3只C57BL/6小鼠注射20 μL 10 mmol/L血红素溶液至纹状体制备成脑出血模型,24 h后采用免疫荧光双标染色检测海马组织冰冻切片上神经元特异性核蛋白(NeuN)和半胱氨酸蛋白酶-3(Caspase-3)的表达。(2)实验二:将原代培养的海马神经元分别用0、10、20、40、80 μmol/L血红素刺激6 h后及用40 μmol/L血红素刺激0、1、2、4、6 h后,采用乳酸脱氢酶(LDH)法检测神经元毒性损伤情况。将原代培养的海马神经元分别用40 μmol/L血红素刺激0、1、2、4、6 h后,采用Western blotting检测神经元中白介素-1(IL-1)受体(IL-IR)1、RIP1、RIP3、MLKL及Caspase-3的蛋白表达,采用酶联免疫吸附法(ELISA)检测白介素-1β(IL-1β)水平。将原代培养的海马神经元分别用0、40 μmol/L血红素刺激6 h后,采用免疫荧光双标染色检测神经元中NeuN和Caspase-3的表达。(3)实验三:将原代培养的海马神经元分别用0、0.5、1.0、1.5 μg/mL IL-IR拮抗剂预孵育1 h后再用40 μmol/L血红素刺激6 h后,采用LDH法检测神经元毒性损伤情况,采用Western blotting检测神经元中IL-IR1、RIP1、RIP3、MLKL的蛋白表达。结果(1)实验一:与假手术组、生理盐水组比较,5 mmol/L血红素组、10 mmol/L血红素组HO-1、RIP1、RIP3、MLKL蛋白表达均明显增高,差异均有统计学意义(P<0.05)。10 mmol/L血红素注射后24 h时的小鼠海马组织冰冻切片上Caspase-3有明显表达,但�ObjectiveTo investigate the effect of hemin on neuronal necroptosis and its mechanism in mice after intracerebral hemorrhage. Methods(1) Experiment one: 24 C57BL/6 mice were divided into sham-operated group, saline control group, 5 mmol/L hemin group, and 10 mmol/L hemin group (n=6);mice in the latter two groups were injected 20 μL 5 mmol/L or 10 mmol/L heme solution into the corpus striatum to prepare cerebral hemorrhage models, and the normal saline group was injected 20 μL normal saline;Western blotting was used to detect the protein expressions of heme oxygenase-1 (HO-1), receptor-interacting protein (RIP)1, RIP3, and mixed lineage kinase domain like protein (MLKL). Three C57BL/6 mice were injected 20 μL 10 mmol/L heme solution into the corpus striatum to prepare cerebral hemorrhage models;24 h after that, double-labelling immunofluorescence was used to detect the expressions of Caspases-3 and neuron-specific nuclear protein (NeuN) in the hippocampus tissues.(2) Experiment two: the primary cultured hippocampal neurons were stimulated with 0, 10, 20, 40 and 80 μmol/L heme for 6 h and 40 μmol/L heme for 0, 1, 2, 4 and 6 h, respectively;lactic dehydrogenase (LDH) method was used to determine the neuron damage. After the primary cultured hippocampal neurons being stimulated with 40 μmol/L heme for 0, 1, 2, 4 and 6 h, respectively, the protein expressions of interleukin-1 receptor (IL-1R)1, RIP1, RIP3, MLKL and caspase-3 in the neurons were detected by Western blotting, and the level of inflammatory factor interleukin-1β(IL-1β) was detected by ELISA. After primary cultured hippocampal neurons being stimulated with 0, 40 μmol/L heme for 6 h, the expressions of NeuN and Caspase-3 in the neurons were detected by double-labelling immunofluorescence.(3) Experiment three: the primary cultured hippocampal neurons were pre-incubated with 0, 0.5, 1.0, and 1.5 μg/mL IL-1R antagonist for one h, and then, stimulated with 40 μmol/L heme for 6 h;the toxic injury of neurons was detected by LDH method, and the protein

关 键 词：脑出血 血红素 神经元 程序性坏死 白介素-1受体 

分 类 号：R743.34[医药卫生—神经病学与精神病学]