局部蛋白质合成和树突可塑性参与度洛西汀镇痛的机制研究

According to our pilot experiment and other publications, subcutaneous injection of formalin into one hindpaw of rat produces persisting ipsilateral mechanical hyperalgesia that lasts for 1-12 days. Laterocapsular division of the central nucleus of the amygdala (CeC) and anterior cingulate cortex (ACC) are two important structures that are closely related to pain. We observed that a pretreatment with duloxetine, a serotonin and norepinephrine reuptake inhibitors (SNRIs), and attenuated formalin induced mechanical hyperalgesia at 24 after injection, which was accompanied with the neuronal activation in the CeC but not spinal dorsal horn. However, how does duloxetine affect the mechanical hyperalgesia remains largely unknown. In vivo studies offer the evidence that serotonin (5-HT) treatment can alter the dendrite structure of the cultured hippocampal pyramidal cells. Thus, we come up with the hypothesis that "single treatment with duloxetine transiently increases the synaptic 5-HT concentration, the increased 5-HT alters the local protein synthesis around the dendritic spines and the changed dendritic structures in CeC and ACC finally alter the nociceptive transmission". To test this hypothesis, we will combined the formalin induced persistent mechanical hyperalgesia model with electron microscope, untrastructural 3-D reconstruction and neuropharmacology techniques to observe the dynamic changes of dendritic spine and local protein synthesis within CeC and ACC, the effect of duloxetine pretreatment on these parameters as well as the potential alterations of duloxetine effects after blocking the local protein synthesis.

我们的预实验和前期研究发现皮下注射福尔马林引起长时程机械性痛敏。中央杏仁核壳部（CeC）和扣带前回（ACC）与疼痛关系密切，我们发现5-羟色胺（5-HT）和去甲肾上腺素（NA）再摄取抑制剂（SNRI）度洛西汀预处理不影响脊髓神经元，但抑制CeC神经元活化并缓解福尔马林所致机械性痛敏，其作用机制不明。离体实验提示5-HT改变ACC神经元树突结构，我们提出假说"单次度洛西汀预处理短暂提高突触间隙5-HT水平，通过快速改变局部细胞骨架蛋白合成，影响CeC和/或ACC神经元树突结构而实现镇痛效应"。本课题拟综合超微形态学、三维结构重塑、神经药理学方法观察福尔马林致长时程机械性痛敏过程中ACC和CeC内局部蛋白质合成和树突结构的动态变化；进而研究度洛西汀预处理对上述指标的影响；最后抑制ACC或CeC部位的局部蛋白质合成，观察其对度洛西汀作用的影响，有望确证我们的科学假说。

在国科金资助下，课题组进行了系列动物实验，发现度洛西汀和赛来西布对小鼠福尔马林痛模型的协同镇痛效应；利用小鼠福尔马林痛模型，进一步观察了重要当归芍药散的镇痛效应及其简单的分子机制；右旋美托咪定和罗匹卡因协同镇痛的可能机制是抑制神经元-星型胶质细胞的交互对话；发现鞘内注射内啡肽-2对卵巢切除大鼠具有强效镇痛效应。这些研究分别提供了以上药物可以用于镇痛治疗的动物实验证据。此外，在本课题资助下，进行了一些循证医学研究，探讨了右美托咪定、抗氧化治疗以及术中输注镁离子制剂对不同疼痛类型的镇痛效应。在本课题资助下共发表标注国科金的国际论文8篇：在包括Molecular Neurobiology等杂志在内的国际期刊上发表4篇研究论著，受本课题资助的其余循证医学研究在J Pain等杂志上发表4篇循证医学论文。但由于核团注射技术比较困难，课题组一直尝试，注射成功率提高后发现药物对局部蛋白合成抑制过低，国科金进行中没有能够完成杏仁核内注射局部蛋白合成抑制剂及其后续实验。今后将在课题负责人承担的其他来源基金的资助下继续完成这部分研究结果。