脊髓多巴胺D1/D2受体复合物调控神经病理性疼痛作用及机制研究

Neuropathic pain is caused by disorders of, or damage to, the nervous system and severely deteriorates the quality of life of 3%-8% of the population. Our previous results indicated that an active component in traditional Chinese medicine, levo-Corydalmine (l-CDL) could antagonize spinal dopamine D1 and D2 receptors to effectively attenuate chronic constriction injury (CCI) induced neuropathic pain, but the mechanism is not clear. Dopamine D1 and D2 receptors couple to Gs and Gi protein respectively, activation of these two G proteins have opposite effects on the excitability of neurons. It has been reported that dopamine D1, D2 receptors can form complexes, the D1/D2 complexes turn out to activate Gq protein to increase the excitability of neurons, activate or antagonize spinal D1 and D2 receptors can increase or decrease the formation of D1/D2 receptor complexes respectively. Our preliminary results indicated that dopamine D1 and D2 receptors were co-expressed in spinal neurons, and in CCI induced neuropathic pain, the co-expression was increased. Administration of l-CDL and dopamine D1, D2 receptor antagonist could decrease the expression of proteins activated in spinal neurons. So we hypothesized that in CCI induced neuropathic pain, dopamine D1 and D2 receptors could form complexes to increase the excitability of neurons, and then promote the development and maintenance of neuropathic pain. l-CDL could antagonize spinal dopamine D1 and D2 receptors to decrease the formation of D1/D2 complexes to remarkably attenuate neuropathic pain.

神经病理性疼痛是指神经系统损伤或功能紊乱引起的疼痛，约3%-8%的人口受到神经痛困扰。我们前期研究显示中药活性成分左旋紫堇达明（l-CDL）能够通过拮抗脊髓多巴胺D1及D2受体缓解CCI诱导的神经病理性疼痛，但D1、D2受体调控疼痛的机制不明确。D1、D2受体分别属于Gs及Gi型G蛋白，单独抑制或激动D1、D2受体对神经元兴奋性具有相反的调控作用。据报道D1、D2受体可形成复合物，转而激活Gq蛋白、IP3受体等增加神经元兴奋性，分别激动（拮抗）D1、D2受体可促进（抑制）此复合物形成。预实验结果显示大鼠脊髓D1、D2受体在神经元中共定位，CCI模型中共定位增多，拮抗D1、D2受体均可缓解CCI诱导的神经痛并抑制脊髓神经元中激活的相关蛋白，因此提出假说脊髓D1、D2受体通过形成复合物，增加神经元兴奋性促进神经病理性疼痛的产生及维持。l-CDL可抑制D1/D2受体复合物的形成发挥强效镇痛作用。

神经病理性疼痛由于其病理机制复杂，临床仍缺乏安全有效的治疗药物。左旋紫堇达明（l-CDL）为中药延胡索中的痕量成分，对其机制研究发现其可拮抗多巴胺D1及D2受体缓解神经病理性疼痛，但多巴胺D1及D2受体分别属于Gαs及Gαi型G蛋白，单独调控D1及D2受体对神经元具有相反的调控作用，而多巴胺D1及D2受体被报道可形成复合物转而激活Gαq蛋白。本项目研究结果显示在神经病理性疼痛模型中脊髓多巴胺D1及D2受体形成复合物增多，抑制D1及D2受体均可抑制复合物的产生。在转染pD1DR-Citrine-N1与pD2DR-mCherry-N1质粒的HEK293细胞中也进一步证实了D1、D2受体及D1/D2受体复合物激动剂可促进复合物的产生，抑制D1、D2受体均可抑制复合物的产生。此外体内研究结果显示多巴胺D1及D2受体通过形成复合物参与调控神经病理性疼痛，即抑制多巴胺D1及D2受体的镇痛作用均可被多巴胺D1、D2受体及受体复合物所取消。体外研究结果显示多巴胺D1及D2受体通过形成复合物增加脊髓神经元兴奋性，即在原代脊髓神经元中，D1、D2受体及受体复合物激动剂均可诱导神经元胞内钙增加，分别联合给予D1及D2受体拮抗剂均可抑制D1、D2受体及受体复合物激动剂诱导的胞内钙离子浓度的增加。进一步体内及体外研究结果显示多巴胺D1及D2受体通过形成复合物调控Gαq，PLC及IP3受体增加胞内钙离子浓度，促进CaMKII，PKCγ，MAPK及CREB的激活进而增加神经元兴奋性促进神经病理性疼痛的产生，而l-CDL可通过拮抗脊髓多巴胺D1及D2受体抑制D1/D2受体复合物的形成抑制脊髓神经元兴奋性缓解神经病理性疼痛。本项目为神经病理性疼痛的治疗提供新的靶标，为阐明l-CDL的镇痛机制提供理论依据，也为临床安全、有效缓解神经病理性疼痛提供先导化合物。