CACNA2D1-CGRP通路调制骨性关节炎痛觉过敏中的效应与机制

One of the important reasons for clinical drug treatment failure is not to relieve effectively osteoarthritis (OA)-induced chronic persistent pain. We found that OA-induced chronic persistent pain was a chronic neuropathic pain rather than a simple inflammatory pain. How to effectively reverse this pain in order to improve clinical drug therapy becomes an urgent problem to be solved. Our preliminary experiments revealed that CACNA2D1 expression were greatly up-regulated in the OA model, while this trend was reversed with CACNA2D1 targeted-inhibitor. It suggests that CACNA2D1 may mediate the occurrence and progression of OA-induced pain. Based on our prior results and latest literature, we speculated that "CACNA2D1-CGRP pathway is involved in modulating OA-induced hyperalgesia" and CACNA2D1-CGRP pathway may be closely related to the occurrence and progression of OA-induced pain. In this study, gene over-expression, RNA interfer and molecular biology techniques etc will be used to tesitify the function and molecular mechanism of CACNA2D1-CGRP pathway in OA-induced pain, as well as animal experiment, to provide a strong evidence and theoretical basis in clinical OA-induced chronic pain treatment and research.

不能有效缓解骨性关节炎（OA）所致的慢性持续性疼痛是临床药物治疗失败的重要原因之一。我们的早期研究证实骨性关节炎（OA）所致的慢性持续性疼痛是一种慢性神经病理性疼痛，而非单纯炎症性疼痛。如何有效逆转该疼痛，以期改进临床药物治疗成为一个亟待解决的问题。我们的预实验揭示：在OA疼痛模型中CACNA2D1表达明显上调，而使用CACNA2D1靶向抑制剂后此种上调被逆转。提示：CACNA2D1很可能介导OA痛的发生及进展。本项目基于既往研究结果和文献分析，提出"CACNA2D1-CGRP通路参与调制骨性关节炎痛觉过敏的效应"的观点，推测CACNA2D1-CGRP途径可能与OA痛发生及进展密切相关。研究采用基因过表达、RNAi等技术，从细胞、组织和动物实验三个水平检测CACNA2D1-CGRP通路在介导OA痛的发生进展中的作用，从而为寻找OA疼痛治疗的新途径、新方法以及为寻找新型药物靶点提供理论依据。

首先，SD大鼠制备成OA模型后处死，取L4,5背根神经节分离背根神经节神经元（DRGs），qPCR和Western blot分别检测正常大鼠及OA模型大鼠DRGs中CACNA2D1的表达情况。构建过表达大鼠CACNA2D1基因的过表达载体，设计并筛选出能有效沉默大鼠CACNA2D1基因的siRNA，并分别瞬时转染低表达CACNA2D1和高表达CACNA2D1基因的DRGs，Western blot检测CGRP、AC、PLC、PKA、PKC、ERK、JNK、p38等信号通路蛋白。其次，30只SD大鼠随机分为6组，分别鞘内注射空载体DRGs、CACNA2D1过表达DRGs、CACNA2D1低表达DRGs、RNAi阴性对照DRGs、CACNA2D1沉默DRGs、CACNA2D1高表达DRGs。鞘内注射2天后，取L4、5背根神经节分离DRGs，提取总RNA及总蛋白，qPCR及Western blot检测DRGs中CGRP及下游通路蛋白AC、PLC、PKA、PKC、ERK、JNK、p38等表达。.CACNA2D1在OA模型中表达明显高于正常大鼠，而CGRP在OA模型大鼠DRGs的表达明显低在正常大鼠；OA模型大鼠的DRGs转染CACNA2D1-siRNAs后，CGRP表达升高，而p-PKA、AC、p-PKC、PLC、p-ERK1/2、p-JNK、p-p38表达降低。正常大鼠的DRGs转染CACNA2D1过表达载体后，CGRP表达降低，而p-PKA、AC、p-PKC、PLC、p-ERK1/2、p-JNK、p-p38表达升高。SD大鼠鞘内注射CACNA2D1过表达的DRGs后，分离纯化的DRGs中CGRP表达较注射空载或CACNA2D1低表达的降低，而CACNA2D1、p-PKA、AC、p-PKC、PLC、p-ERK1/2、p-JNK、p-p38表达升高；而SD大鼠鞘内注射CACNA2D1沉默的DRGs后，分离纯化的DRGs中CGRP的表达较注射RNAi阴性对照DRGs或CACNA2D1高表达DRGs的升高，而而CACNA2D1、p-PKA、AC、p-PKC、PLC、p-ERK1/2、p-JNK、p-p38表达降低。.研究结果表明， CACNA2D1-CGRP 通路在介导OA 痛的发生进展中起着重要的作用，从而为寻找OA 疼痛治疗的新途径、新方法以及为寻找新型药物靶点提供理论