脊髓背角兴奋性突触跨突触信号Neurexin1β/Neuroligin1在术后疼痛中的作用及机制研究

Postoperative pain is commonly seen in clinical practice and the neurobiological mechnisms underlying remain unclear. Our previvous studies demonstrate the critical roles of spinal AMPA receptor GluR1 subunits trafficking and synaptic targeting in central sensitization of postoperative pain. However, the upstream signaling is still elusive.Based on the pilot and previous studies, we hypothesized that transsynaptic signaling of Neurexin1β/Neuroligin1 was involved in postoperative pain through modulating the release of excitatory neurotransmitters and the PSD-95/stargazing mediated synaptic targeting of spinal AMPA receptor GluR1. To test the hypothesis, we will examine the effects of Neurexin1β/Neuroligin1 on presynaptic Ca2+-dependent release of excitatory neurotransmitters and postsynaptic targeting of spina AMPA receptor GluR1 subunits, by using immunoblot, gene silencing, spinal microdialysis, immunoprecipitation, molecular cloning, expression purification of fusing protein, etc. We also will construct a cell-permeable fusing peptide, which is used to disrupt the interaction between PSD-95 and stargazing, and test its analgesia action. The proposal will reveal novel mechanisms underlying postoperative pain and will provide some new targets for pain treatment...

外科手术组织损伤是临床上最常见的急性疼痛，其神经生物学机制仍不太清楚。我们前期的研究证实，脊髓背角AMPA受体GluR1亚基转运和突触靶向是术后疼痛脊髓中枢敏化的重要机制，但启动这一过程的上游信号不明。基于预实验结果和前人的研究我们推测脊髓背角兴奋性突触跨突触信号Neurexin1β/Neuroligin1参与术后疼痛的中枢敏化。为验证此假说，我们将采用免疫印迹、基因沉默、脊髓微透析、免疫共沉淀、分子克隆、融合蛋白表达与纯化等技术和手段，研究术后疼痛期间跨突触信号Neurexin1β/Neuroligin1对突触前Ca2+依赖性兴奋性神经递质释放的影响以及该信号对突触后AMPA受体亚基突触靶向的影响，并将构建细胞通透性融合肽，干预Neuroligin1与PSD-95的相互作用，检测其镇痛效应。本项目揭示术后疼痛的脊髓新机理，为镇痛药物的研发提供新靶点。

Neuroligin1是一种突触细胞粘附分子， 可通过蛋白间的相互作用参与到突触功能的调节。然而其在术后疼痛中的作用仍不清楚。本研究选取健康清洁成年雄性SD大鼠，通过建立切口痛模型模拟术后疼痛；分别在术前，术后3小时，1d，和3d测试行为学，包括累计疼痛评分和机械痛阈，之后取脊髓背角进行实验。使用western方法研究切口痛术后Neuroligin1、GluR1、GluR2和PSD-95在脊髓背角突触后膜表达的变化，使用免疫荧光方法研究Neuroligin1与PSD-95在突触后膜的定位；使用免疫共沉淀方法研究切口痛术后Neuroligin1与PSD-95共表达的变化；同时使用Neuroligin1的siRNA抑制Neuroligin1的表达，观察Neuroligin1被抑制后疼痛相关受体GluR1在脊髓背角突触后膜的表达变化，以及Neuroligin1被抑制后对Neuroligin1和PSD-95相互作用的影响。结果显示切口痛可以导致大鼠脊髓背角突触后膜Neuroligin1和GluR1的表达增加，同时Neuroligin1与PSD-95的相互作用增加。而使用siRNA抑制Neuroligin1的表达后，与盐水组相比，大鼠的累计疼痛评分显著降低，机械痛阈显著增加。大鼠脊髓背角突触后膜疼痛相关受体GluR1也相应的被抑制，同时Neuroligin1与PSD-95的相互作用也被抑制。本研究认为抑制Neuroligin1的表达可以通过抑制突触后膜Neuroligin1和PSD-95的相互作用，同时抑制GluR1的突触靶向来抑制术后疼痛，魏术后疼痛的治疗提供了新的靶点。