触液核ATP-P2X4Rs信号通路在电针百会穴治疗神经病理性痛中的机制研究

Electroacupuncture at peripheral acupoints could be used for the treatment of neuropathic pain, and the involved mechanism was associated with the spinal nociceptive pathway. Our preliminary results found that electroacupuncture at Baihui attenuated the hyperalgesia induced by nerve damage, while the mechanisms remains elusive. Previous reports indicated that ATP-P2X4Rs pathway in cerebrospinal fluid-contacting nucleus (CSF-CN) play critical role in the pathogenesis of neuropathic pain, and ecto-5’-nucleotidase (NT5E) which hydrolyze ATP to adenosine are involved in the regulation of these processes. In addition, electroacupuncture could increase the activity of NT5E through the activation of the adrenergic nerve system. Based on these, we render such hypotheses: electroacupuncture at Baihui could increase the NT5E activity in brain, decrease ATP levels, resulting in the deactivation of P2X4Rs in CSF-CN ultimately lead to the attenuation of neuropathic pain. In these research, the chronic constriction injury rat model will be used to observe the effect of electroacupuncture at Baihui on ATP-P2X4Rs in CSF-CN, NT5E levels, and pain. The agonist or antagonist, and overexpression or knockdown of ATP-P2X4Rs will be used to elucidate the mechanisms of the treatment effect produced by electroacupuncture at Baihui. These work will reveal the roles of ATP-P2X4Rs in neuropathic pain with electroacupuncture at Baihui and provide the theory basis for its wide clinical use.

以往研究显示针刺外周穴位治疗神经病理性痛（NP）与调控脊髓水平的伤害感受性通路有关。我们前期研究发现电针头部百会穴也可缓解神经损伤后的痛觉过敏，但机制不明。中脑触液核ATP-P2X4Rs信号通路在NP的发生和发展中起关键作用，并受ATP水解酶5’-外核苷酸酶（NT5E）调控。鉴于电针可通过激活肾上腺素能神经通路，增加NT5E的活性，我们提出假说：触液核ATP-P2X4Rs信号通路是电针百会穴治疗NP的关键靶点，即电针通过增强脑内NT5E活性，降低ATP水平，减少P2X4受体激活，缓解NP。本项目拟采用坐骨神经结扎大鼠模型，观察电针对触液核ATP-P2X4Rs、NT5E和疼痛的影响，通过激动/拮抗剂、基因过表达或敲减等干预手段，明确触液核ATP-P2X4Rs信号通路在电针百会穴治疗NP中的作用，旨在从脊髓上中枢水平揭示电针百会穴治疗NP的机制，为拓展针刺百会穴的临床应用范围提供理论依据。

背景 神经病理性痛（NP）是一种难治性的疼痛状态，目前机制不明。以往研究显示脊髓小胶质细胞上嘌呤受体P2X4Rs的表达在NP调控中起到极其重要的作用。中脑中央导水管周围灰质（PAG）和触液核（CSF-CN）是中枢内源性镇痛系统的重要组成部分。本项目研究触液核上P2X4Rs表达在慢性坐骨神经结扎损伤（CCI）模型大鼠疼痛调控中的作用以及电针百会穴缓解NP的可能机制。.方法 制备CCI模型，应用行为学实验、侧脑室注射技术、免疫组化以及蛋白印迹实验、萤火虫荧光素酶ATP检测等手段，观察大鼠痛阈变化，明确触液核ATP的浓度变化以及P2X4Rs表达水平。予以电针百会穴干预，采用核团注射工具药干预ATP和P2X4Rs，观察模型鼠行为学和P2X4Rs表达的变化。.结果 P2X4Rs表达在正常大鼠PAG和触液核上。模型鼠痛阈在造模后第7天最低，触液核上P2X4Rs的表达呈上升趋势。在造模后第2、3、5、7、10、14天，病毒注射组和电针组的机械缩足阈值（MT）与CCI组的差异均有统计学意义。电针后，模型鼠MT在造模后第2、3、5、7、10、14天升高；与CCI组相比，触液核P2X4Rs表达和ATP浓度显著下调。模型鼠行电针治疗，分别正、负干预ATP后，Apyrase组与CCI组、α,β-meATP组与电针组的MT均有差异，P2X4Rs表达在电针组与α,β-meATP组、Apyrase组与α,β-meATP组均有差异；分别正、负干预P2X4Rs后，与IVM组相比，电针组和5-BDBD组的MT均有差异。（P<0.01）.结论 嘌呤受体P2X4Rs在大鼠PAG神经元和触液核上表达。触液核P2X4Rs表达参与脑干水平神经病理性疼痛的中枢调控。敲减触液核P2X4Rs可以逆转模型鼠疼痛的发生。电针百会穴衰减模型鼠的疼痛程度，触液核的ATP-P2X4Rs信号通路参与此调控。本项目从脊髓上中枢水平研究触液核上P2X4Rs表达在NP调控中起到极其重要的作用，有助于进一步探索NP的发生机制，同时为拓展针刺百会穴的临床应用范围提供理论依据。