基于原代培养背根神经元兴奋性研究东亚钳蝎毒素治疗神经性疼痛的物质基础以及作用靶点

Neuropathic pain is a common phenomenon in the clinic. Although the mechanisms are diverging, the abnormal neuronal firing is thought to be the converging point responsible for the neuropathic pain. Therefore, inhibition of the neuron excitability may confer useful analgesic strategy. The Chinese Scorpion (Buth Martensii Karch) is widely used in clinic to treat several diseases especially neuropathic pain.We and others have discovered several analgesic peptides from the crude venom, however, the analgesic effect of these pure peptides is much less potent and less efficacious than the crude venom, warranting the significance of further investigating the analgesic components in the scorpion venom. We therefore hypothesize that some peptides with strong analgesic effect have not been discovered, or different analgesic peptides in the crude venom which can interact with distinct sites within the same receptor or the sites between the distinct receptors or signaling pathways can act synergistically or in an additive manner to relieve the pain. We therefore aim to 1: Systematically investigate the analgesic peptides of scorpion venom and their molecular targets based on their inhibition of the neuron excitibility; 2: Investigate the analgesic effect of distinct analgesic peptides either alone or in combination; 3: Examined the molecular targets and the selectivity of the scorpion peptides on various subtypes of receptors. To fulfill the goals, we will establish a novel rapid throughput model using primary cultured spinal cord neurons to detect the influence of the scorpion toxins on the network excitability to guide the purification and structure elucidation which will be performed by using HPLC, Edman degradation as well as various biochemical methods. We will also examine the inhibitory effect on the network excitability of the scorpion peptides in singly or in combination to evaluate their synergistic, additive or antagonistic effect. The molecular targets of the analgesic peptide will be examined in primary cultured neurons using various pharmacological and biochemical tools or in the cell lines heterologously expressed specific subtype of the ion channels/ receptors. Finally, the analgesic effect of scorpion peptides in singly or in combination will be confirmed in animal models. With our state of the art pharmacological platform coupled with latest peptide purification and identification technologies, we believe that we can elucidate the analgesic components in the scorpion crude venom and clarify their molecular targets. Furthermore, investigating analgesic effect of different combination of scorpion analgesic peptides permits us to discover better combination of scorpion peptides on the manipulation of pain. Moreover, the rapid throughput platform we established in the project will facilitate the discovery of analgesic lead compounds from Chinese Traditional Medicine and elucidation of their molecular targets and mechanisms.

神经性疼痛在临床发病广泛，虽其发病机制多样，但神经元异常放电是多种疼痛机制的汇聚点。东亚钳蝎在临床上用于治疗神经性疼痛等疾病，疗效确切。但目前分离到的多肽的镇痛活性都比粗毒弱。我们推测：蝎毒中存在镇痛活性更强的多肽或不同多肽的镇痛效果具有协同或叠加效应。本课题拟建立以背根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多肽；研究不同活性多肽的叠加或协同作用；同时利用表达受体亚型的细胞系,研究镇痛多肽的作用靶点及受体选择性。最后对能够抑制神经兴奋性的多肽用体内疼痛模型评价其疗效。本研究将建立一个全新的快速高效镇痛药物筛选模型；阐明蝎毒镇痛的物质基础,作用靶点，及不同多肽镇痛的叠加或协同作用，为治疗神经性疼痛提供新结构、新策略；也将发现受体特异性高的多肽用于研究受体结构和功能；同时也为防治神经性疼痛的中药物质基础及作用靶点的研究提供新的技术平台。

神经性疼痛危害严重，机制复杂。背根神经元作为痛觉传入的第一级神经元，在疼痛感觉的传导过程中起着极为重要的作用。东亚钳蝎作为传统中药之一，有息风镇痉、通络止痛、攻毒散结之功效。本课题在国家自然科学基金的资助下，建立了背根神经元神经网络钙振荡模型，并利用化合物对钙振荡的影响，利用多种技术从东亚钳蝎蝎毒中分离得到并鉴定了17个毒素的序列，以及21个分子量新颖的毒素；利用钙振荡模型对蝎毒素及天然产物进行了筛选，发现了一系列天然小分子化合物，譬如苦木碱庚（DHCT），大花紫玉盘醇类化合物（UGs）以及小部分蝎毒毒素能够抑制神经网络钙振荡，在动物CCI神经性疼痛模型上，DHCT和UGD能够显著抑制神经性疼痛。研究中发现部分蝎毒素能够增加神经元的内钙水平，作用机制研究发现其能够延缓钠离子通道的失活过程，在小脑颗粒细胞能够引起神经元死亡，然而此类毒素在背根神经元细胞具有神经营养作用，能够刺激神经突的生长，可能与神经元的修复相关；另一类蝎毒毒素能够增加背根神经元的钙振荡频率和振幅，抑制钾离子通道，对背根神经元具有神经营养作用，刺激神经突的生长，并在动物CCI神经性疼痛模型上显著抑制疼痛。此研究工作表明，蝎毒素对疼痛的治疗作用可能是通过蝎毒素的神经营养作用，从而修复神经元，产生镇痛效果。同时本研究建立了多个表达不同离子通道亚型的细胞系，并建立了相应的快速发现靶点特异性配体的研究方法，为研究化合物的靶点特异性提供了研究平台。本研究建立了一个全新的快速高效镇痛药物筛选模型，依据此模型能够快速发现神经系统活性物质，同时依据化合物对钙振荡影响的表型，可以初步确定化合物的作用靶点；利用此方法，发现了具有神经系统活性的蝎毒毒素和天然小分子化合物，初步阐明了蝎毒的物质基础以及部分毒素的作用靶点。本研究将为研究防治神经性疼痛的中药物质基础及作用靶点提供新的技术平台，同时有望发现治疗神经性疼痛的先导化合物。