两种罂粟科植物镇痛活性成分的发现及作用机制研究

Neuropathic pain is one of the most widespread and intractable human complaints. Although the mechanisms of neuropathic pain are complicated, the abnormal neuronal firing is thought to be a converging point. It has proven to be an useful strategy to manage neuropathic pain by inhibiting neuronal excitability. The tubers of Corydalis decumbens (Thunb.) Pers. and Dactylicapnos scandens (D. Don) Hutch. are widely used in clinical for the treatment of neuropathic pain with definite therapeutic effect. We will establish a novel rapid throughput model using primary cultured spinal cord dorsal horn neurons to detect the influence of crude components or single compounds on the network excitability. The analgesic constituents from the two medicinal plants and their molecular targets will be systematically investigated guided by their inhibitory effects on the neuron excitibility. The structure-activity relationships of the analgesic compounds will be studied based on the pharmacological results in conbination with structure modificatuion.The molecular targets of the active compounds will be examined in primary cultured neurons using various pharmacological and biochemical tools or in the cell lines heterologously expressed the ion channels/receptors. Finally, the analgesic effect of active compounds will be evaluated in animal models. The aim of this project is to study the material foundation and action mechanisms of the two herbs, clarify fundamental scientific principle that they are used for the treatment of pain, provide academic and experimental gist for their clinical applications, and develop new research methods and models for the exploitation of innovative analgesic drugs from abundant Chinese herbs and natural plant resources.

神经病理性疼痛在临床发病广泛且难以治愈，虽其发病机制多样，但神经元异位放电所导致的过度兴奋是多种疼痛机制的汇聚点，抑制神经元的兴奋性已经被证明具有镇痛作用。夏天无和紫金龙临床上广泛应用治疗神经性疼痛，疗效确切。本课题拟建立以脊髓背角神经元兴奋性为指标的快速体外疼痛模型，并以其为导向,利用多种技术分离并鉴定能够抑制神经元兴奋性的化合物，结合结构改造和修饰探讨活性化合物的构效关系。同时利用表达与疼痛相关受体/通道的细胞系,研究镇痛化合物的作用靶点。最后对能够抑制神经兴奋性的活性化合物用体内疼痛模型评价其疗效。本项目拟通过药效物质基础、作用机制等方面的系统研究，阐释两种药材治疗疼痛的科学内涵，阐明其干预疼痛的路径，为这两个品种的临床推广应用提供重要的理论和实验依据，并探索一套以中药为源头开发镇痛创新药物可借鉴的模式和方法。

神经病理性疼痛发病广泛，严重影响了患者的身体健康和生活质量，目前缺乏有效的治疗药物。广泛存在于罂粟科植物中的异喹啉类生物碱是镇痛活性先导化合物的可靠来源，已经从中开发出了比如吗啡、延胡索乙素、青藤碱等药物。罂粟科药用植物夏天无和紫金龙临床应用于疼痛的治疗历史悠久、疗效确切，对其药效物质基础研究有望能发现镇痛先导化合物。目前已从夏天无和紫金龙中分离并鉴定了120个生物碱成分，其中新化合物32个，生物碱的结构类型为异喹啉类生物碱。对分离得到的生物碱单体化合物进行体外镇痛活性的筛选，发现一个苯酞异喹啉化合物Corydecumine G（Cor G）可以浓度依赖性的抑制脊髓背角神经元的自发钙振荡，提示Cor G对神经元的兴奋性有较好的抑制作用。鉴于Cor G体外活性实验在微摩尔浓度下可抑制神经元兴奋性，本项目以egenine这一大量成分为起始原料进行化学半合成获得了足量该化合物。用坐骨神经结扎（CCI）诱导的小鼠神经病理性疼痛模型评价了该化合物的体内镇痛活性。研究结果发现Cor G能够显著增加大鼠患侧足底的机械刺激阈值和热刺激缩足时间，表明能够缓解神经性疼痛症状。利用膜片钳技术来考察Cor G对疼痛相关离子通道的影响，发现化合物可以抑制钠通道开放，从而抑制脊髓神经元去极化，进而抑制钙振荡，抑制脊髓神经元兴奋性，抑制神经元细胞的异常放电，缓解神经元敏化从而改善神经性疼痛。本项目还通过体内和体外实验考察了Cor G对小胶质细胞活化的影响。研究结果发现Cor G可以通过降低CCI动物模型中脊髓组织及活化的BV2小胶质细胞中MAPK信号通路磷酸化水平，调节CCI大鼠脊髓组织和活化的BV2细胞中炎症因子含量，抑制小胶质细胞活化。综上所述，Cor G可通过中枢和外周双重途径调控神经病理性痛疼，具有较好的研究开发前景。此外，还发现egenine具有显著的抗肾纤维化活性。研究成果共计发表SCI论文11篇，部分论文在Organic Letters、Journal of Natural Products、Bioorganic Chemistry、Phytochemistry、Journal of Chemical Neuroanatomy等国际知名期刊上发表，并申报相关专利二项。