钙通道α2δ1亚基调控延髓5-HT3A受体功能介导慢性头面部疼痛机制研究

Orofacial pain is a debilitating disorder that diminishes the quality of life and seriously inhibits the health of the patient. Trigeminal pain also spreads to wide orofacial areas and is a serious clinical problem. It has been well known that primary orofacial pain signal is firstly processed by subnucleus caudalis (Vc) of the spinal trigeminal nucleus. Chronic constriction injury of the infraorbital nerve (CCI-ION) caused orofacial hypersensitivity that correlated with voltage-gated calcium channel (VGCC) α2δ1 subunit (Cavα2δ1) up-regulation in trigeminal ganglion neurons and Vc. Blocking Caα2δ1 with gabapentin, a ligand for Caα2δ1 proteins led to a reversal of orofacial hypersensitivity, supporting an important role of Caα2δ1 in orofacial pain processing. However, the corresponding mechanisms are still unclear..It has been proposed that upregulation of Cavα2δ1 in sensory neurons of dorsal root ganglion (DRG) and dorsal spinal cord by peripheral nerve injury has been suggested to contribute to neuropathic pain. The Vc is considered to have similar anatomy and function with the spinal cord. Combining the findings from Vc and the spinal cord, we propose that there is a potential circuits involved in the development of neuropathic pain. It is as follows: Peripheral nerve injury induced Cavα2δ1 upregulation in trigeminal ganglia (TG) leads to increased Cavα2δ1 translocation to the dorsal cord pre-synaptic terminals through the central axons of sensory neurons. This results in increased pre-synaptic excitatory input to activate postsynaptic NK1 receptor expressing neurons in Vc, leading to activation of spinal-supraspinal descending serotonergic facilitatory pathway located in rostral ventromedial medulla (RVM). This in turn can activate 5-HT3A receptors in Vc. .To investigate this hypothesis, we have created transgenic mice, including Cre-LoxP mice and mice that constitutively overexpress Cavα2δ1 in neuronal tissues. Using behavioral testing, qPCR, Western Blot, immunohistochemistry and in vitro electrophysiological recording, we are trying to address the following issues: 1) Whether the activation of Cavα2δ1 contributes to the development of chronic orofacial pain？2) Whether this effect depends on the activation of 5-HT3A receptors mediating the descending facilitation from RVM? 3) What is the main location of 5-HT3A receptors that mediates the Cavα2δ1-involved effects, excitatory interneurons or afferent fiber terminals? Together, this study aims to provide new evidence for the mechanisms of chronic orofacial pain and new sight for its treatment.

抑制三叉神经脊束核尾侧亚核（Vc）内电压门控钙通道α2δ1亚基（Cavα2δ1）功能显著缓解慢性头面部疼痛，其机制尚不明确。结合脊髓水平的研究，我们提出如下假说：神经损伤后，三叉神经节（TG）和Vc内Cavα2δ1表达增加，激活介导延髓头端腹内侧区（RVM）5-羟色胺（5-HT）能下行易化作用的5-HT3A受体，引发慢性头面部疼痛。既往研究特异性差。本研究利用Cre-Loxp系统条件性敲除或过表达等方法建立的转基因小鼠，采用痛行为学测试、qPCR、Western blot、免疫组化及膜片钳等方法，重点解决以下问题：1）Cavα2δ1是否参与神经损伤后慢性头面部疼痛的发生？2）该效应是否通过激活介导RVM的5-HT能下行易化作用的5-HT3A受体？3）介导该效应的5-HT3A受体的细胞定位：兴奋性中间神经元还是TG初级感觉神经元的中枢端末梢？旨在阐明Cavα2δ1参与慢性头面部疼痛的机制。

三叉神经痛表现为骤发骤停的剧烈疼痛，且极易扩散至临近区域，引发大面积难以忍受的慢性疼痛，且临床病人表现为对冷刺激敏感，其治疗极具挑战性，研发有效镇痛、不易耐受且副作用无或小的药物具有极其重要的意义。初级感觉神经元，如三叉神经节（TG）神经元是痛觉信息传递的第一站，便于外周给药，可以作为药物作用的重要靶点。Cavα2δ1是电压门控钙通道（VGCC）的亚单位之一，参与痛觉信号转导。研究表明，三叉神经节神经元中枢端末梢的Cavα2δ1可能参与慢性头面部疼痛的发生，然而其是否参与继发痛及机制尚不明确。.本研究建立三叉神经第二支分支眶下神经结扎后切断的小鼠三叉神经痛模型（pT-ION），采用痛行为学测试、病毒注射干扰或过表达、qPCR、Western blot、免疫组化及细胞培养等方法，对Cavα2δ1是否参与三叉神经损伤所致原发痛和继发痛及分子机制进行了初步探索。主要结果如下：（1）pT-ION诱发稳定的原发痛和继发痛，且可被Cavα2δ1拮抗剂加巴喷丁GBT缓解；（2）Cavα2δ1在三叉神经节及三叉神经脊束核尾侧亚核（Vc）表达上调，且与三种类型神经元标志物CGRP、IB4及NF200均共标；（3）神经损伤后在TG和Vc p-PKC、TRPA1和GJ表达均显著上调；（4）特异性敲减TG Cavα2δ1的表达，显著缓解pT-ION诱发的PKC-TRPA1表达上调及原发痛和继发痛；（5）过表达Cavα2δ1诱发三叉神经病理痛及PKC-TRPA1/GJ信号通路的表达上调，TRPA1或GJ拮抗剂显著缓解过表达Cavα2δ1诱发的三叉神经病理痛；（6）PKC抑制剂显著缓解pT-ION及Cavα2δ1过表达所致痛敏行为，逆转TRPA1和GJ的表达上调。.综上所述，本研究显示，PKC-TRPA1/GJ信号通路介导Cavα2δ1参与三叉神经痛原发痛和继发痛的发生。本研究提示，Cavα2δ1及下游PKC-TRPA1/GJ信号通路中蛋白分子是三叉神经损伤所致机械及冷刺激过敏和扩散治疗的重要靶点。