针刺调控P2X3受体介导的PKC/NMDA信号通路缓解干眼眼表神经痛的分子机制研究

干眼并伴有神经痛是临床常见眼表疾病，其药物治疗面临困境。针刺有效缓解患者干眼神经痛，但其机制尚不清楚。课题组前期观察到针刺下调感觉神经通路中P2X3受体表达并缓解干眼神经痛。本项目将重点探讨针刺如何调控P2X3受体所介导疼痛信号转导，发挥其抑制干眼神经痛效应和机制。为此基于临床有效穴位，用针刺治疗干眼兔模型，采用神经示踪技术观察针刺作用角膜感觉神经通路的关键部位，从中分析针刺对神经元P2X3受体及其介导的PKC/NMDA信号通路关键基因和蛋白表达；利用细胞膜片钳技术观察针刺调控P2X3受体介导的离子通道电流密度；有针对性选用P2X3受体拮抗/激动剂，探寻针刺对疼痛信号转导变化，阐述针刺调节P2X3受体及其介导的PKC/NMDA疼痛信号通路，提高角膜知觉阈值，缓解干眼神经痛的分子机制。这些创新工作将明确针刺调控角膜感觉神经传导通路干预干眼神经痛新途径，有望对针刺治疗干眼神经痛产生突破性进展。

Dry eye disease (DED) with neuropathic pain is a common ocular surface disease. The treatment with drug is facing difficulties. Acupuncture can effectively relieve ocular neuropathic pain in DED patients, but the underlying mechanism is not clear. Our previous experiments observed that acupuncture could downregulate P2X3 receptor expression in sensory nerve pathway as well as relieve neuropathic pain in DED. This project will mainly explore the inhibitory effects and mechanisms of acupuncture on neuropathic pain in DED by regulating P2X3 receptor and its-mediated PKC/NMDA pain signal transduction. The rabbit model of DED will be treated with acupuncture according to the clinical effective acupoint. We will observe the key sites for acupuncture acting on the corneal sensory nerve pathway by neural tracing technique, and then analysis the effects of acupuncture on the expression levels of key genes and proteins in P2X3 receptor-mediated PKC/NMDA signaling pathway. Moreover, we will use whole-cell patch-clamp technique to observe the current density of P2X3 receptor-mediated voltage-gated ion channel mediated by acupuncture. On the other hand, P2X3 receptor antagonist and/or agonist will be respectively used to explore the changes of acupuncture on pain signal transduction. This project will elaborate the molecular mechanisms by which acupuncture relieves neuropathic pain in DED via regulating P2X3 receptor and its-mediated PKC/NMDA pain signal pathway to increase corneal sensation threshold. These novel finding will clarify a new way to regulate corneal sensory nerve pathway in the intervention of dry eye neuropathic pain by acupuncture, and be expected to make a breakthrough in the treatment of dry eye neuropathic pain with acupuncture.