红车轴草异黄酮抑制小胶质细胞活化保护多巴胺能神经元的研究  

Trifolium pratense isoflavones protect dopaminergic neurons by inhibiting microglia activation

作　　者：陈寒青[1] 孙汉巨[1] 汪锡金[2] 徐学明[3] 金征宇[3] 

机构地区：[1]合肥工业大学生物与食品工程学院,安徽合肥230009 [2]上海交通大学医学院附属瑞金医院神经内科,神经病学研究所,上海200025 [3]江南大学食品科学与技术国家重点实验室,食品学院,江苏无锡214122

出　　处：《中国药理学通报》2011年第3期390-396,共7页Chinese Pharmacological Bulletin

基　　金：合肥工业大学博士专项基金项目(No2010HGBZ0293)

摘　　要：目的探讨从红车轴草分离的5种异黄酮芒柄花素(formononetin)、大豆黄素(daidzein)、毛蕊异黄酮(calycosin)、红车轴草素(pratensein)、德鸢尾素(irilone)对脂多糖(LPS)诱导的多巴胺能神经元损伤的保护作用。方法利用LPS建立多巴胺能神经元损伤的细胞模型,利用液闪测定法检测[3H]DA摄取力以评价多巴胺能神经元的功能,利用免疫细胞化学技术进行酪氨酸羟化酶(tyrosine hydroxylase,TH)染色观测多巴胺能神经元的形态和数目,利用免疫细胞化学技术进行OX-42染色观测小胶质细胞的形态和数目。NO测定依据Griess反应,TNF-α测定采用酶联免疫法,超氧自由基测定采用细胞色素C还原法。结果从红车轴草分离的5种异黄酮均以剂量依赖方式减轻大鼠中脑原代混合培养体系中LPS诱导的多巴胺摄取力的下降和多巴胺能神经元数目的下降。而且,这5种异黄酮还抑制中脑混合培养体系和小胶质细胞纯化体系中LPS诱导的小胶质细胞的活化和TNF-α、NO和超氧自由基的生成。并且,这5种异黄酮作用强弱的顺序依次为:pratensein>daidzein>calycosin>formononetin>irilone。结论从红车轴草分离的5种异黄酮对LPS诱导的多巴胺能神经元的损伤均具有不同程度的保护作用,且保护作用强弱的顺序依次为:pratensein>daidzein>calycosin>formononetin>irilone,抑制小胶质细胞的活化是其作用机制之一。Aim To explore the protective effect of five isoflavones（formononetin,daidzein,calycosin,pratensein,irilone） from Trifolium pratense on lipopolysaccharide（LPS）-induced dopaminergic neurodegeneration.Methods An in vitro model of LPS-induced dopaminergic neurodegeneration was used to investigate the protective effect of five isoflavones from Trifolium pratense on dopaminergic neurons against LPS-induced neurotoxicity by measuring theDA uptake and counting of TH-immunoreactive cells.Microglia were visualized by staining for the CR3 complement receptor with monoclonal antibody OX-42.The production of nitric oxide（NO）was determined by measuring the accumulated level of nitrite in the culture supernatant with the Griess reagent,and the release of tumor necrosis factor-α（TNF-α）was measured with a rat TNF-α enzyme-linked immunosorbent assay kit.The production of superoxide was determined by measuring the superoxide dismutase（SOD）-inhibitable reduction of cytochrome C.Results All five isoflavones dose-dependently attenuated LPS-induced decrease in dopamine uptake and the number of dopaminergic neurons in rat mesencephalic neuron-glia cultures.Moreover,they also significantly inhibited LPS-induced activation of microglia and production of TNF-α,NO and superoxide in rat mesencephalic neuron-glia cultures and microglia-enriched cultures.In addition,the potency order of these five isoflavones was：pratenseindaidzeincalycosinformononetinirilone.Conclusions All five isoflavones from Trifolium pratense may protect dopaminergic neurons from LPS-induced injury and their effects in inhibiting microglia activation may be one of the mechanisms.Moreover,the rank order of protective potency of these five isoflavones is：pratenseindaidzeincalycosinformononetinirilone.

关 键 词：红车轴草 异黄酮 小胶质细胞 多巴胺能神经元 肿瘤坏死因子α 一氧化氮 超氧自由基 

分 类 号：R284.1[医药卫生—中药学] R322.8[医药卫生—中医学]