基于蛋白质组学探讨安神定志方干预抽动障碍大鼠的作用机制

Mechanism of Anshen Dingzhi Fang in Tic Disorder Rat Model： A Proteomics-Based Investigation

    摘要
  • 摘要:
    目的 评价安神定志方对3，3'-亚氨基二丙腈（3，3'-Iminodipropionitrile， IDPN）诱导的抽动障碍（Tic disorders， TD）大鼠模型的治疗效果，并利用定量蛋白质组学技术，探讨其干预TD大鼠纹状体蛋白质表达的分子机制。
    方法 选用雄性SD大鼠，通过腹腔注射IDPN建立TD模型。成功造模后，将大鼠随机分为模型组，硫必利组和安神定志方低、中、高剂量组，并设空白组，连续灌胃给药28 d。评估大鼠的刻板行为；采用酶联免疫吸附试验（Enzyme-linked immunosorbent assay，ELISA）检测血清中多巴胺（Dopamine， DA）、5-羟色胺（5-Hydroxytryptamine， 5-HT）、肿瘤坏死因子-α（Tumor necrosis factor-α， TNF-α）和白细胞介素-6（Interleukin-6， IL-6）的水平；采用苏木精-伊红（Hematoxylin and eosin staining，HE）染色观察纹状体组织的病理学改变。应用串联质量标签（Tandem mass tag，TMT）定量蛋白质组学技术结合平行反应监测（Parallel reaction monitoring，PRM）靶向验证，分析安神定志方对TD大鼠纹状体蛋白质组的影响。
    结果 与模型组相比，安神定志方能显著改善TD大鼠的刻板行为（P<0.05），降低大鼠血清中升高的DA、5-HT及TNF-α水平（P<0.05），并改善IDPN所致的纹状体神经元紊乱、皱缩、水肿及胶质细胞增生的病理损伤。TMT技术共鉴定到179个在安神定志方中剂量组与模型组间差异表达的蛋白。基因本体论（Gene ontology，GO）和京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes， KEGG）通路富集分析显示，这些差异蛋白显著富集于多巴胺能突触、谷氨酸能突触信号传递。安神定志方能显著逆转与囊泡运输相关的关键蛋白（Rab3a、Rab3gap2、Arf3）的异常表达，且该结果通过PRM技术得到靶向验证。
    结论 安神定志方对IDPN诱导的TD大鼠模型具有治疗作用，其作用机制涉及调节神经递质平衡、抑制神经炎症反应以及保护纹状体神经元等。

     

    Abstract:
    OBJECTIVE To evaluate the therapeutic effects of the traditional Chinese medicine (TCM) formula, Anshen Dingzhi Fang (ASDZF), in a rat model of tic disorders (TD) induced by 3,3'-Iminodipropionitrile (IDPN), and to elucidate the underlying molecular mechanisms by examining its effects on the striatal proteome using quantitative proteomics.
    METHODS Male Sprague-Dawley (SD) rats were used to establish a TD model via intraperitoneal injection of IDPN. Following successful model establishment, the rats were randomly assigned to a model group, a tiapride group, and low-, medium-, and high-dose ASDZF groups. A normal control group was also included. All intervention groups received continuous oral gavage for 28 days. Stereotyped behaviors were assessed. The serum levels of dopamine (DA), 5-hydroxytryptamine (5-HT), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA). Pathological changes in the striatum were observed using hematoxylin and eosin (HE) staining. Tandem mass tag (TMT)-based quantitative proteomics, combined with parallel reaction monitoring (PRM) for targeted validation, was applied to analyze the effects of ASDZF on the striatal proteome of TD rats.
    RESULTS Compared with the model group, ASDZF significantly ameliorated stereotyped behaviors (P<0.05), reduced the elevated serum levels of DA, 5-HT, and TNF-α (P<0.05), and mitigated the IDPN-induced pathological damage in the striatum, including neuronal disorganization, pyknosis, edema, and glial proliferation. TMT analysis identified 179 differentially expressed proteins (DEPs) between the medium-dose ASDZF group and the model group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these DEPs were significantly enriched in pathways related to the dopaminergic synapse and glutamatergic synapse. ASDZF significantly modulated the expression of key proteins associated with vesicular transport, including Rab3a, Rab3gap2, and Arf3, which was subsequently validated by PRM.
    CONCLUSION ASDZF exerts therapeutic effects on the IDPN-induced TD rat model. Its mechanism of action is multi-targeted and involves multiple pathways, including the regulation of neurotransmitter balance, inhibition of neuroinflammatory responses, and protection of striatal neurons.

     

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