钩吻素子抗神经病理性疼痛的脊髓TSPO作用靶点研究

The study on new targets of drug action for the prophylaxis and treatment of neuropathic pain is to be undertaken urgently. The translocator protein (18 kDa) (TSPO) in glia cells of spinal cord may become a new therapeutic target for neuropathic pain. Our previous study has shown that koumine possesses the significant antagonistic effect on neuropathic pain, which may be concerned with the agonism of TSPO in spinal cord. The present project is undertaken to further confirm that the antagonistic action of koumine on neuropathic pain is related to the agonism of TSPO in spinal cord. Based on this, firstly, from point of view about affinity and agonism effect, it is to be explored whether koumine is through the direct activation of spinal TSPO to exert the antagonistic effect on neuropathic pain. By using chemical proteomics approaches, it is to be confirmed whether TSPO is a target for koumine action. Subsequently, from an in vitro cellular system and an in vivo animal level, it is to be analyzed that koumine regulates the function of TSPO on the production of neuroactive steroids. Finally, it is to be investigated that the molecular pathway underlying TSPO mediating the analgesic effect of koumine against neuropathic pain, so as to elucidate the spinal molecular mechanism about the action of koumine against neuropathic pain. This project is undertaken to offer the new target and pathway of drug action for research and development of new prophylactic and therapeutic drugs against neuropathic pain, to contribute to a better understanding of the central mechanism of neuropathic pain, and to establish foundation for proposing the new efficiency of koumine against neuropathic pain.

防治神经病理性疼痛(NPP)新的药物作用靶点亟待研究。脊髓神经胶质细胞内TSPO有望成为治疗NPP药物新的作用靶标。我们前期工作表明，钩吻素子具有显著的抗NPP作用，并且可能与脊髓TSPO激动有关。本项目在明确钩吻素子抗NPP作用与脊髓TSPO激动有关的基础上，首先拟从亲和力与激动效应角度，探索钩吻素子可否直接激动脊髓TSPO发挥抗NPP作用；接着用化学蛋白质组学技术确认TSPO是否为钩吻素子的作用靶点；然后在细胞水平与整体水平上，分析钩吻素子对TSPO介导神经活性甾体生成之功能的影响，以期较为全面地提出TSPO作为钩吻素子作用靶点的科学依据；最后探讨脊髓TSPO介导钩吻素子抗NPP作用的分子通路以及关键效应分子，阐释钩吻素子抗NPP作用的脊髓分子机制。本研究将为防治NPP新药研发提供新的药物作用靶点与途径，并有助于深化理解NPP的中枢机制；也为提出钩吻素子抗NPP新功效奠定理论基础。

防治神经病理性疼痛新的药物作用靶点亟待研究。脊髓神经胶质细胞内TSPO有望成为治疗神经病理性疼痛药物新的作用靶标。本项目分别在大鼠Ⅰ型、Ⅱ型糖尿病性神经病理性疼痛模型上，首次观察到钩吻素子对糖尿病性神经病理性疼痛具有显著的治疗作用，拓展了钩吻素子抗神经病理性疼痛作用谱。在大鼠CCI模型上，采用荧光免疫组织化学法观测了神经病理性疼痛发生发展过程中大鼠脊髓背角TSPO表达的动态变化以及钩吻素子对之作用，发现神经病理性疼痛发生发展过程中大鼠脊髓背角术侧TSPO表达持续增强，钩吻素子可显著抑制其TSPO的表达。分析了TSPO特异性拮抗剂PK11195对钩吻素子抗神经病理性疼痛效应的拮抗作用，明确了钩吻素子抗神经病理性疼痛作用与脊髓TSPO的激动有关；而CCI模型大鼠其脊髓背角TSPO表达增强，给予钩吻素子反而抑制其表达，提示存在钩吻素子直接靶向TSPO而发挥抗神经病理性疼痛作用。采用放射配体结合分析法，于国内首次发现钩吻素子可正性别构调节TSPO，钩吻素子可能为TSPO的正性别构调节剂，TSPO可能是钩吻素子的重要作用靶点。在细胞水平与整体水平上，本研究观察到钩吻素子可显著提高神经活性甾体3α,5α-四氢孕酮（别孕烯醇酮）的水平，提示钩吻素子可促进TSPO介导的神经活性甾体的生成，并与其发挥抗神经病理性疼痛作用相关。系列研究表明，脊髓TSPO介导钩吻素子抗神经病理性疼痛作用的分子通路以及关键效应分子为钩吻素子→脊髓TSPO→上调3α,5α-四氢孕酮生成→激动GABAA受体→抗神经病理性疼痛，阐释了钩吻素子抗神经病理性疼痛作用的脊髓分子机制。上述研究成果为防治神经病理性疼痛新药研发提供新的药物作用靶点与作用方式，并有助于深化理解神经病理性疼痛的中枢机制；也为提出钩吻素子抗神经病理性疼痛新功效奠定理论基础。