COX2-PGE2-EP信号通路在大鼠背根节损伤后根性神经痛发生中的作用机制及芪麝丸对其调控的效应机

The pathogenesis of neuropathic pain is that disc herniation or the compression of vertebral body osteophyte to nerve tissue can cause inflammation, then induce the excessive excitement of cervical dorsal root ganglion and ectopic activity , resulting in pain. Therefore, The inflammation reaction of DRG and its peripheral nerve tissue is an important pathologic basis of root pain , the pain cascade mediated by COX2-PGE2-EPsignaling pathways plays a prominent role in the generation of radicular pain following the nerve injury..The project team follows the theory of syndrome differentiation and treatment of traditional Chinese medicine ,that is"qi is main and blood is first",which is proposed by shi department of traumatology,they put forward " YiQiHuaYu therapy " is the rule of the treatment of the radicular pain and develop a new drug of Chinese herbal medicine named Qishewan (It is composed of astragalus membranaceus 15 g, ligusticum wallichii 12 g, artificial musk 0.01 g, artificial bezoar 3 g, caulis sinomenii 9 g, prevent has 9 g, etc) for radicular pain . Previous studies have shown that the drug can inhibit animal model’s activity of inflammatory mediators of radicular pain obviously ,such as PGE2, substance P,meanwhile it can also improve microcirculation. But the function link of how to alleviate the inflammatory response of nerve root needs further research. Clearing the function key link of Qishewan and revealing its mechanism of alleviating inflammation are helpful to the development of traditional Chinese medicine ’s "qi and blood theory" and also lay the foundation of further improvement of clinic efficacy.

椎间盘突出或者椎体骨赘压迫神经根引起炎症反应，诱发背根神经节过度兴奋和异位活动，从而产生疼痛，是根性神经痛的发病机制。因此，DRG及其周围神经组织的炎症反应是根性痛的重要病理基础，COX2-PGE2-EP1信号通路介导的外周途径和COX2-PGE2-EP4信号通路介导的中枢途径在根性神经痛的产生中具有重要作用。.项目组遵循石氏伤科“以气为主，以血为先”的中医理论，提出了以“益气化瘀法”为主治疗根性神经痛的法则，开发出治疗中药新药芪麝丸（由黄芪15g、川芎12g、人工麝香0.01g、人工牛黄3g、青风藤9g、防己9g等组成）。前期研究显示，该药明显抑制神经根压迫动物模型神经根炎症介质PGE2、P物质活性，改善微循环。但如何缓解颈神经根炎症反应的作用环节有待深化研究。明确芪麝丸作用的关键环节，揭示芪麝丸缓解根性神经痛的作用机制，有助于发展中医“气血理论”，为该药的进一步临床应用奠定理论基础。

目的：以COX2-PGE2-EP信号通路为切入点，深入探讨芪麝丸对背根神经节损伤后COX2-PGE2-EP 信号通路的影响，明确芪麝丸作用的关键环节，确定芪麝丸的有效成分，进一步阐述该芪麝丸缓解根性神经痛的作用机制。. 方法：（1）建立大鼠腰神经根慢性压迫模型，分别于造模后3、7、14、21、28天应用芪麝丸进行灌胃干预1个月，观察压迫损伤和药物干预后大鼠热刺激和机械刺激行为学变化，采用RT-PCR法、Western Blot 法、LC-MS/MS 技术等检测不同时间压迫损伤以及芪麝丸干预后COX2-PGE2-EP信号通路相关基因、蛋白表达变化，以及芪麝丸在大鼠血清中药物浓度变化。（2）体外培养背根神经元细胞，应用TNF-α诱导炎性环境，应用芪麝丸有效组分进行干预，观察芪麝丸有效组分干预后COX2-PGE2-EP信号通路相关基因、蛋白表达变化。. 结果：（1）芪麝丸可以显著提高背根节压迫损伤后大鼠机械性刺激痛阈值，延长热刺激回缩时间。压迫损伤后，COX2-PGE2-EP信号通路COX2、PGE2、EP4和TNF-αmRNA表达及EP1、NOS蛋白表达均显著升高，其随时间变化与大鼠行为学变化趋势相同；芪麝丸可以显著降低COX2、PGE2、EP4和TNF-αmRNA表达和EP1、NOS蛋白表达。（2）芪麝丸有效组分青藤碱可显著减少TNF-α可诱导体外培养背根节神经元背根元凋亡，改善神经元形态，降低COX2、PGE2、EP4mRNA表达，对EP1mRNA表达无明显作用。（3）芪麝丸干预可诱导压迫损伤后背根神经元555个基因上调，1151个基因下调。结合测序基因变化选取细胞自噬信号通路核心蛋白检测显示，压迫损伤后pAkt、mTOR蛋白显著升高，芪麝丸干预和显著降低pAkt蛋白和mTOR蛋白表达。. 结论：芪麝丸可以通过抑制COX2-PGE2-EP信号通路中COX2、PGE2、EP1和EP4基因和蛋白表达，到达减缓背根神经元压迫损伤后炎性根性神经痛的发生；芪麝丸的有效组分青藤碱主要对COX2-PGE2-EP4信号通路有明显抑制作用，对COX2-PGE2-EP1信号通路无明显作用；芪麝丸对自噬信号通路核心蛋白pAkt、mTOR蛋白具有明显的抑制作用，提示在该药物在调节细胞自噬方面存在重要的研究价值。