D2R-Cx43通路调控镜像痛敏的分子与细胞互作机制

Single side of somatic tissue or neuropathic injuries often result in pain hypersensitive responses displayng on bilateral side of injury, which is named as mirror image pain. However, induction and modulation mechanism underlying this kind clinical pain is still far from clear. The dopamine D2 receptor (D2R), gap junction intercellular communication and glia participate in the regulation of pain through different manners. Recently, our new findings indicated that crosstalk of D2R-Cx43 signal pathway mediated mirror image pain under pathological pain conditions. In the present project, with the advantages of our newly-established pain model mimicking experimental scorpion sting, the mirror image pain-involved plastic expression, modulation of D2R, Cx43 and its phosphorylation in bilateral spinal and dorsal root ganlion would be investigated by using animal behavioral observation, pharmacological, electriphysiological, molecular and cellular biological techniques from wild and D2R knockout and Cx43 conditional knockout animals. The elucidation of molecular and cellular interactive mechanism of D2R-Cx43 pathway underlying mirror-image pain would greatly promote our understanding of pain mechanisms, and surely provide some science and practical supports for development of new strategies for diagnosis, therapy and analgesics of clinical pain.

躯体单侧组织或神经损伤可导致损伤对侧同源性健侧体节出现所谓“镜像痛敏”，其发生机制仍有待阐明。在多巴胺D2受体（D2R）、缝隙连接通道蛋白Connexins、神经胶质细胞参与疼痛调制的不同途径,我们幸运地新发现，D2R-Cx43间存在互作调控的信号通路新方式。本项目拟综合应用动物行为学、药理学、电生理学、分子和细胞生物学等手段，利用项目组建立的实验性蝎蛰镜像痛敏模型，分别检测野生型、D2R和Cx43敲除型动物单侧外周损伤后脊髓、背根神经节双侧D2R、连接蛋白Cx43及其磷酸化表达和调控的可塑性变化与镜像痛敏发生、发展和维持的相关性，以阐明D2R-Cx43通路介导镜像痛敏的分子与细胞互作机制，推动对疼痛相关缝隙连接通道的细胞环路整合调控的系统化认识，为临床慢性痛诊治新策略研发提供新的科学和实验基础。

躯体单侧组织或神经损伤不仅导致损伤部位表现持续自发痛、原发性痛敏和损伤部位周围区域继发性痛敏，还可导致损伤部位对侧对称部位出现所谓“镜像痛敏”。本项目即立意于镜像痛敏发生和持续慢性化机制研究，基于项目组建立的实验性蝎蛰镜像痛敏模型，综合应用行为药理学、qRT-PCR、免疫荧光、免疫印迹、活细胞成像等方法，重点探索单侧外周损伤后脊髓、背根神经节双侧多巴胺D2受体（D2R）、缝隙连接蛋白Cx43及其磷酸化表达和调控的可塑性变化与镜像痛敏发生、发展和维持的相关性，意图进一步理解D2R与Cx43通路调控镜像痛敏的分子细胞内源性机制，取得的系列重要研究成果和新发现如下：.（1）单侧疼痛刺激后，双侧脊髓、DRG缝隙连接蛋白Cx43呈不对称性的时程和细胞差异表达分布，并以通道蛋白的开放、表达量改变等多种方式参与镜像痛敏的发生、发展和维持。脊髓Cx43 Ser262、Tyr265、Ser279/282、Ser368与DRG Cx43 Ser368位点磷酸化的差异性激活，PKC/PKA蛋白激酶在此过程中至关重要。.（2）阻断D2R降低痛刺激诱发的Cx43表达上调或激活，阻断Cx43降低痛刺激诱发的D2R的上调或激活，并在脊髓星形胶质细胞、DRG卫星胶质细胞表现为D2R激活可开放缝隙连接通道或半通道。表明D2R与缝隙连接通道Cx43之间存在互相调控机制。.（3）D2R敲除降低脊髓、DRG和卫星胶质细胞Cx43的表达。多巴胺刺激显著增高卫星胶质细胞Cx43的表达，而缝隙连接阻断剂逆转之，表明DRG卫星胶质细胞D2R对Cx43表达和功能性开放有重要的正反馈调控效应。.综上，在动物脊髓和DRG整体、星形胶质细胞、小胶质细胞、卫星胶质细胞确立了D2R-PKA/PKC-Cx43表达、磷酸化、功能性开放的正反馈信号调控通路，为疼痛，尤其为镜像痛敏发生、发展和维持的分子细胞机制提供了新的理解。