慢性束缚大鼠海马差异蛋白质表达及逍遥散干预作用

Differentially Expressed Proteins in Rat Hippocampus after Chronic Immobilization Stress and Intervention Using Xiao Yao San Decoction

  • 摘要:
    目的分析肝郁脾虚证模型大鼠海马组织蛋白质的差异表达, 从蛋白质组学角度探讨肝郁脾虚证的实质,同时研究逍遥散对肝郁脾虚证模型大鼠海马组织差异蛋白质的调节作用。
    方法SD大鼠24只, 按体重随机分为正常对照组、7天模型组、21天模型组、21天模型+逍遥散组,每组各6只。以慢性束缚应激法建立肝郁脾虚证大鼠模型, 运用双向凝胶电泳(2-DE)方法检测各组大鼠海马差异表达蛋白质并采用western blot方法验证部分海马差异表达蛋白质。
    结果双向凝胶电泳分析发现17个具有显著差异表达的蛋白质斑点, 经鉴定确认的8个蛋白质分别为:胶质纤维酸性蛋白-2(GFAP-2)、微管蛋白α-1c(Tubulin α-1c)、细胞质肌动蛋白2、14-3-3蛋白、β-2a微管蛋白、磷脂酰乙醇胺结合蛋白(PEBP)、突触核蛋白α-syn3以及低分子量蛋白(18KDa protein)。筛选出的8个差异表达蛋白质, 在肝郁脾虚证大鼠海马组织中的6个蛋白质表达上调。1个蛋白质下调,还有一个蛋白质五种不同的亚型有不同的上下调结果。其功能主要涉及免疫、信号转导、细胞周期、细胞凋亡、酶活性调节细胞骨架,突触可塑性等方面。Western blot方法检测的两个差异表达的蛋白质中,胶质纤维酸性蛋白-2(GFAP-2)与差异蛋白质检测结果吻合,突触核蛋白α-syn3的检测结果条带模糊,但依然能通过灰度比值证实2D电泳结果的可靠性。
    结论肝郁脾虚证模型大鼠海马存在差异表达蛋白质,其发生发展是多种蛋白质参与的结果,其生物学功能涉及突触可塑性、信号转导、细胞周期、细胞凋亡、酶活性调节、细胞骨架、信号通路等方面。逍遥散能调节差异蛋白质表达水平,从而发挥重要的调控作用。

     

    Abstract:
    ObjectiveTo identify differentially expressed proteins in the hippocampus of rats after chronic immobilization stress (CIS) using a proteomics approach, and to study the effect of the Xiao Yao San (XYS) decoction on differentially expressed proteins.
    MethodsTwenty-four Sprague Dawley rats were randomly assigned to one of four groups of equal body weight: control (non-stress), 7-day stress, 21-day stress and 21-day stress + XYS treatment groups. Two-dimensional gel electrophoresis (2-DE) was used to detect differences in protein expression in rat hippocampus. One differentially expressed protein was measured and verified by western blotting.
    ResultsSeventeen proteins showed differential expression. Among these, eight could be identified: glial fibrillary acidic protein-2 (GFAP-2), tubulin alpha-1c, cytoplasmic muscle actin 2, 14-3-3 protein, β-2a tubulin, phosphatidylethanolamine binding protein, synuclein α syn3, and a low molecular weight (18 kD) protein. Six of these proteins exhibited increased expression, one showed decreased expression, and the other protein, which comprised five subtypes, were either increased or decreased. These proteins are known to be involved in immunity, signal transduction, cell cycle control, apoptosis, regulation of enzyme activity, cytoskeleton structure, and synaptic plasticity. GFAP-2 was further analyzed, and its differential expression confirmed by western blotting.
    ConclusionSome proteins are differentially expressed in the hippocampus of rats under chronic stress. The biological functions of these differentially expressed proteins are varied. Finally, the XYS decoction can significantly up- or down-regulate these protein expression levels.

     

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