缝隙连接及ERK/MAPK信号系统在激痛点所致肌筋膜疼痛中的作用及机制研究

Myofascial trigger points (MTrPs), defined as hyperirritable nodules of spot tenderness in a muscle taut band, have been suggested to be the common cause of myofascial pain syndromes (MPS). However, current understanding of the pathophysiology of MTrPs is incomplete. Current evidence suggests that spinal cord sensitization contribute to the localized and generalized mechanical hyperalgesia associated with MTrPs. Nevertheless，the detailed mechanisms of spinal sensitization in MTrP-related pain are lacking. The gap junction (GJ) and connexin 43(Cx43) have been shown to play an important role in chronic neuropathic pain and in the initiation and maintenance of spinal cord sensitization. The phosphorylation of Cx43 can be modulated by ERK / MAPK pathway. MTrPs as peripheral sources of noxious inputs may sensitize primary and secondary sensory neurons and may share similar sensitization mechanisms as neuropathic pain. We thus hypothesize that GJ and Cx43 may be involved in the MTrP-related pain and spinal sensitization may be regulated by the ERK / MAPK signaling system. To test this hypothesize, we plan to investigate the effects of GJ and Cx43 on MTrP-related pain and the regulatory effects of ERK / MAPK on Cx43 in rat spinal cord dorsal horn. The methods of freeze-fracture electron microscopy, dye tracer technique, Western blot, RT-PCR, Cx43 transfection, siRNA interference, two-cell bipolar patch clamp, and laser scanning confocal microscopy would be used in this project aimed at finding new molecular targets in chronic musculoskeletal pain conditions associated with MTrPs.

激痛点（MTrP）的存在是肌筋膜疼痛综合征难以彻底治疗的主要原因之一，目前MTrP的病理基础还未得到充分证实。我们和其他学者的前期研究均提示脊髓敏化参与MTrP痛觉增敏，但机制尚未明确。缝隙连接（GJ）及其连接蛋白Cx43在慢性疼痛及脊髓敏化的形成和维持上有重要作用。ERK/MAPK能调节多种细胞Cx43磷酸化。因此，我们推断：GJ及Cx43在ERK/MAPK信号系统调节下参与MTrP疼痛及脊髓敏化。本项目拟通过动物实验，从组织和细胞水平采用电镜冷冻蚀刻、染料示踪、Western blot、RT-PCR，Cx43转染、siRNA干扰等技术明确脊髓背角GJ及Cx43在MTrP疼痛中的作用及ERK/MAPK信号系统对脊髓胶质细胞Cx43的调节作用；再采用双细胞双极膜片钳和激光共聚焦动态研究ERK/MAPK信号系统对GJ电导和通讯作用的影响。这将为MTrP的治疗提供新靶点，找到缓解疼痛的有效方法

本课题组在基金项目规定的时间顺利完成了全部计划。实验中不断进行文献查新补充，在原有实验计划基础上，我们增加了高渗盐水激痛点组，以进一步验证激痛点对应的脊髓后角的各种变化。我们选取自然产生激痛点和tant band的大鼠，在其股二头肌的潜在MTrP注射小量谷氨酸将其活化，取脊髓背角和鞘内注射GJ阻滞剂甘珀酸及星形胶质细胞抑制剂后，分别从脊髓背角组织和星形胶质细胞培养水平采用电镜冷冻蚀刻、染料示踪、Western blot、RT-PCR、激光共聚焦、siRNA干扰、Cx43转染等技术明确脊髓背角星形胶质细胞间GJ在MTrP疼痛和SEA中作用及ERK/MAPK信号系统中相关激酶和磷酸酶对Cx43蛋白的基因和蛋白磷酸化水平的调节作用。双细胞双极膜片钳和激光共聚焦动态研究ERK/MAPK信号系统对GJ电导和通讯功能的影响及相邻细胞间GJ开放速率部分实验不理想。原因为因项目主要负责人出国进修一年，导致时间紧张，膜片钳技术高，难以掌握。已进行的实验及研究结果已经说明和充分提示本研究计划的最初设想，活性MTrP大鼠脊髓背角存在GJ及脊髓敏化，GJ、 ERK/MAPK信号系统参与激痛点疼痛和SEA过程。这将为肌筋膜疼痛综合征及慢性骨骼肌肌肉疼痛疾病的治疗研究提供新的靶点，找到缓解疼痛的有效方法。