从脊髓背角Apelin-APJ途径研究电针调控神经-胶质细胞网络抗慢性炎性痛的分子机制

Apelin/angiotensin receptor-like protein J receptor (APJR) signaling is present in the neurvous system and peripheral tissues and plays a critical role in modulating multiple aspects of physiology and pathophysiology. This study is designed to investigate the effect of electroacupunture (EA) on Apelin/APJR signaling in a rat model of complete Freund's adjuvant-induced chronic inflammatory pain, while the acupoints of Yanglingquan (GB34) and Zusanli (ST36) are used. The distribution and expression change of Apelin and APJR in different neural and glial cell types in spinal dorsal horn will be examined after EA treatment in rat with inflammatory pain. Furthermore, the activity of three pathways of Cyclic Advenosine Monophosphato (cAMP)/protein kinase A (PKA), phospholipase C (PLC)/ protein kinase C (PKC) and phosphatidylinositol 3-kinase PI3K/AKT, which are the three mainly downstream signaling pathways of Apelin-APJR signaling, will be examined that whether they have cell type special regulations by EA stimulation in this pathological condition. Importantly, we focus our attention on how EA-induced Apelin-APJR signaling regulation could reciprocally affect the different activity of these pathways in different cell types and communicate the neural and immune systems in chronic inflammatory pain management. The different cell types following different strategies to response to EA stimulation study could offer a new aspect for us to understand the traditional theory of acupuncture systematically therapeutic effects. This study would potentially help clinicians to further understand the mechanism underling the analgesic effects of EA on pain management and also identify whether the Apelin-APJR system is a potential therapeutic target in chronic inflammatory pain.

以佐剂诱导大鼠慢性炎性疼痛模型，电针针刺大鼠"阳陵泉"和"足三里"穴，在观察电针抗炎镇痛效应的基础上，系统观察电针对慢性炎性痛大鼠腰段脊髓背角神经肽Apelin和受体血管紧张肽受体样蛋白质受体（angiotensin receptor-like protein J receptor，APJR）的影响，分析Apelin/APJR通路在腰段脊髓背角神经元区域分布特征。同时采用神经药理学等多种手段，观察电针对病理条件下Apelin/APJR通路下游信号转导通路cAMP/PKA、 PLC/PKC和PI3K/AKT在神经元和胶质细胞不同细胞类型中区域特异性活性的影响。重点观察电针如何通过Apelin/APJR通路影响和沟通神经元-胶质细胞中不同的下游信号传导通路活性，调节神经-免疫相互作用而产生协同效应，揭示针灸治疗慢性炎性痛调控细胞信号转导的新原理，为临床有效治疗慢性炎性痛解决关键科学基础问题。

电针广泛应用于各种病理性疼痛的治疗，而慢性炎性痛是针刺治疗有效病种之一。然而，针刺效应机制并未完全阐明。以往的研究表明，侧脑室注射或鞘内注射apelin-13不仅显著提高正常小鼠的疼痛阈值，而且对内脏疼痛或炎症性疼痛有明显的镇痛作用。但是内源性的Apelin/APJ系统在慢性疼痛中的分子机制尚不清楚。本研究组发现，电针刺激后可显著上调高反应大鼠脊髓APJ的mRNA表达，提示APJ的表达可能与电针镇痛密切相关。因此，我们⑴利用弗氏佐剂诱导大鼠慢性炎性疼痛模型（CFA 模型），研究模型大鼠腰段脊髓背角在神经元和胶质细胞apelin 及其受体APJ mRNA和蛋白表达变化的规律；⑵Apelin/APJ 通路在电针镇痛效应中的作用；⑶电针对Apelin/APJ 通路下游信号的调节规律。结果发现，⑴弗氏佐剂注射后可引起大鼠显著的机械痛敏和热痛敏，而电针刺激可显著改善炎性痛；⑵apelin 及其受体APJ在腰段脊髓背角神经元特异性表达，而星形胶质细胞和小胶质细胞均无表达；⑶弗氏佐剂诱导大鼠慢性炎性疼痛后，模型组大鼠腰段脊髓背角apelin及其受体APJ的mRNA和蛋白减少，电针处理后能显著抑制apelin和APJ的表达下调；⑷鞘内给予Apelin/APJ系统拮抗剂F13A能抑制电针抗痛觉过敏效应；⑸电针可显著抑制慢性炎性痛大鼠PKA催化亚基α（PKAca）、磷酸化环腺苷酸反应元件结合蛋白（p-CREB）、p-PKCε、p-PI3K和p-Akt的表达增加，但是采用F13A干预，发现仅翻转了电针对PKAca和p-CREB的抑制作用；⑹外源性鞘内给予apelin-13可增强电针的镇痛效应；⑺血浆apelin因子可能与临床电针治疗膝关节骨性关节炎的疗效相关。通过本研究，明确了⑴脊髓背角Apelin/APJ 系统表达于神经元，参与了慢性炎性痛的形成和发展；⑵脊髓背角Apelin/APJ 系统及其下游信号通路CAMP/PKA介导了电针治疗慢性炎性痛效应的产生；⑶针联合鞘内给予apelin-13治疗慢性炎性痛可发挥协同效应。本研究揭示针灸治疗慢性炎性痛调控细胞信号转导的新机制，为临床有效治疗慢性炎性痛提供了科学依据。