脊髓背角KA受体GluK2亚基功能调节及与AMPA受体GluR1亚基交叉谈话机制在术后疼痛中作用及干预研究

The intrathecal administration of classic AMPA/KA receptor antagonists has shown potent analgesic action. However, the side effects limit their clinical use. Since the selectivity of classic AMPA/KA receptor antagonists for KA receptor is much higher than that for AMPA receptor, KA receptor maybe plays a critical role in spinal nociception during incisional pain. The latest studies have demonstrated the unique functional regulations of KA receptors. It remains unclear whether the functional regulations of KA receptors are involved in pain hypersensitivity. Based on our pilot studies, we hypothesized that the interaction of KA receptor GluK2 subunits with NETO2, GRIP, through enhancing the kinetics of ion channels and synaptic targeting, and the cross-talk between KA receptor and AMPA receptor were implicated in incisional pain hypersensitivity. To test the hypothesis, by using PCR, Western blot, immunohistochemistry, immunoprecipitation, fusing protein expression and purification, and electrophysiology, we would investigate ①the GluK2 mRNA editing, trafficking and synaptic targeting; ② the effects of the interaction of GluK2-NETO2, GluK2-GRIP on GluK2 desensitization, trafficking and synaptic targeting; ③the mechanism underlying the cross-talk between KA receptor and AMPA receptor during incisional pain. Our project may reveal the new mechanisms of postoperative pain and find some new analgesic targets.

术后鞘内给予经典型AMPA/KA受体拮抗剂有很强的镇痛效应，但副作用妨碍了临床使用。由于经典型AMPA/KA受体拮抗剂对KA受体的选择性相对较高，提示KA受体在术后痛敏中有更重要作用。最新研究发现，KA受体具有独特的功能调节机制，然而这些功能调节过程是否参与痛觉敏化尚不清楚。基于预实验结果，我们推测术后脊髓背角KA受体GluK2亚基可与NETO2、GRIP蛋白相互作用改变通道特性和实现突触富集，并与AMPA受体交叉谈话，促进术后痛敏。为验证此假说，我们将采用PCR、免疫组化、免疫印迹、免疫共沉淀、钙离子成像、融合肽表达纯化和电生理等技术，研究术后① GluK2 mRNA编辑、胞浆-胞膜转运和突触靶向的变化；②GluK2-NETO2、GluK2-GRIP相互作用对KA受体通道特性、转运和突触靶向的影响；③KA受体与AMPA受体的交叉谈话机制。该项目将揭示术后疼痛的新机制，发现新的镇痛靶点。

术后急性疼痛是临床上最常见和最需要即刻解决的一类疼痛。急性术后疼痛控制不佳会导致慢性疼痛的发生，降低患者生活质量，增加医疗负担。既往研究发现，鞘内给予经典型AMPA/KA受体拮抗剂有很强的镇痛效应，但副作用妨碍了临床使用。由于经典型AMPA/KA受体拮抗剂对KA受体的选择性相对较高，提示KA受体在术后痛敏中有更重要作用。最新研究发现，KA受体具有独特的功能调节机制。在本项目中，我们对脊髓背角KA受体参与术后疼痛脊髓中枢敏化的功能调控机制进行了深入研究。我们的研究发现：① 切口痛刺激可诱发脊髓背角神经元KA受体GluK2亚基 mRNA未编辑形式增加，从而增加KA受体对Ca2+通透性，有助于术后疼痛脊髓信号的转导；②切口疼痛刺激可诱发脊髓背角神经元KA受体GluK2亚基发生胞浆-胞膜转运和突触靶向的变化，突触后膜GluK2与GRIP发生相互作用在突触后膜富集，参与伤害性信号的脊髓突触传递；敲减GRIP的表达能明显抑制GluK2-GRIP蛋白-蛋白相互作用，抑制GluK2突触后膜富集，从而抑制疼痛敏化；③ 切口痛刺激通过增加脊髓背角神经元突触后膜上GluK2-GRIP相互作用实现对AMPA受体的交叉谈话，抑制GluK2-GRIP相互作用可抑制AMPA受体GluR1亚基的突触后膜转运，而对GluR2亚基转运没有影响，最终使突触后膜上对Ca2＋通透性AMPA受体增加，脊髓背角神经元兴奋性增加，从而增加了伤害性信号的突触传递效能，有利于痛觉脊髓中枢敏化；④切口手术后，切口周围皮肤角质生成细胞发生增殖和表达IL-1beta和TNF，糖尿病小鼠切口周围皮肤角质生成细胞增殖和表达IL-1beta和TNF较正常小鼠切口更持久，且疼痛行为的表现也较正常小鼠更长。该项目揭示了术后疼痛的新机制，丰富了对切口痛脊髓中枢敏化的机制认识，发现了多个术后疼痛治疗的新靶点。对未来开发针对性的镇痛药物提供了新启示。