单核细胞趋化因子-1调控NMDA-NR2B在激痛点所致脊髓敏化中的作用及机制研究

Myofascial trigger points (MTrPs), defined as hyperirritable nodules of spot tenderness in muscle taut bandS, 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. CCL2/monocytes chemoattractant protein-1 has been shown to play an important role in chronic neuropathic pain and in the initiation and maintenance of spinal cord sensitization through regulating astrocyte-neuron interaction. N-methyl-D-aspartic acid receptor-NR2B of neurons also has a very closely relationship with neuropathic pain and central sensitization. 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 CCL2 and CCR2(main receptor of CCL2) may be involved in the MTrP-related pain and spinal sensitization through regulating astrocyte-neuron interaction and the NMDA-NR2B recptor activity. To test this hypothesis, we plan to investigate the effect and mechanism of CCL2/CCR2 on MTrPs-related spinal cord sensitization through regulating NMDA-NR2B activity in rat spinal cord dorsal horn. The methods of electromyography, pain-related behavior, Western blot, RT-PCR, siRNA interference, laser scanning confocal microscopy and whole cell patch clamp technique would be used in this project aimed at finding new molecular targets in chronic musculoskeletal pain conditions associated with MTrPs.

激痛点（MTrP）是肌筋膜疼痛综合征难以彻底治疗的主要原因，其病理基础未得到充分证实，尚缺乏有效治疗方法。我们和其他学者的研究均提示MTrP对应脊髓存在中枢敏化，胶质细胞功能增强，神经元体积变小，但具体机制尚未明确。单核细胞趋化因子CCL2及其受体CCR2通过介导胶质细胞–神经元相互作用参与中枢敏化和慢性疼痛调节。谷氨酸受体NMDA-NR2B和神经病理性疼痛及中枢敏化密切相关。因此我们推断CCL2/CCR2通过介导胶质细胞–神经元相互作用调控NMDA-NR2B参与MTrP所致的脊髓敏化。本项目拟通过动物实验，从组织、细胞及功能水平采用肌电图、行为学、Western blot、RT-PCR、激光共聚焦、siRNA干扰，全细胞膜片钳明确CCL2/CCR2在MTrP疼痛、自发性电活动及脊髓敏化中作用，其对NMDA-N2B的调节作用及机制。这将为MTrP治疗提供趋化因子方面的新靶点。

激痛点（MTrP）是肌筋膜疼痛综合征难以彻底治疗的主要原因，其病理基础未得到充分证实，尚缺乏有效治疗方法。我们和其他学者的研究均提示MTrP对应脊髓存在中枢敏化，胶质细胞功能增强，神经元体积变小，但具体机制尚未明确。单核细胞趋化因子CCL2及其受体CCR2通过介导胶质细胞–神经元相互作用参与中枢敏化和慢性疼痛调节。谷氨酸受体NMDA-NR2B和神经病理性疼痛及中枢敏化密切相关。本课题组2016年申请到国家自然基金“单核细胞趋化因子-1调控NMDA-NR2B在激痛点所致脊髓敏化中的作用及机制研究”。.在该课题的支持下，课题组在基金项目规定的时间顺利完成了全部计划，大部分实验工作进展和结果很好。.我们选取自然产生激痛点和taut band的大鼠，在其股二头肌的潜在MTrP注射小量谷氨酸或高渗盐水将其活化，鞘内注射CCR2的拮抗剂RS504393及Cx43拮抗剂Gap19和ERK1/2拮抗剂 SCH772984后，取脊髓背角，从动物行为学、组织水平免疫荧光、Western blot、RT-PCR等技术，研究活性MTrP大鼠脊髓背角CCL2/CCR2的改变及其在MTrP疼痛动物行为学及SEA中的改变。从细胞水平，采用星形胶质细胞-神经元共培养、分别加入CCR2激动剂/拮抗剂，CCR2激动剂+Cx43拮抗剂，CCR2激动剂+ERK1/2拮抗剂，和分别进行Cx43siRNA/CCR2siRNA/ERK1/2siRNA干扰，Western blot技术，RT-PCR检测技术及激光共聚焦显微镜进行NMDA-NR2B，CREB，P-CREB，P-Tyr1472- NR2B及钙离子的测定等。也进行了全细胞膜片钳记录神经元NMDA电流实验，这部分实验进展不顺利，记录到电流不稳定。.已进行的实验及研究结果（行为学、组织水平和细胞水平等）充分说明和提示本研究计划的最初设想，活性MTrP大鼠脊髓背角存在CCL2/CCR2，CREB，P-CREB，NR2B、 Cx43、ERK1/2表达增加，鞘内注射CCR2拮抗剂，大鼠疼痛和SEA改变，上述蛋白表达减少,CCL2/CCR2参与MTrP疼痛、SEA及脊髓敏化；CCL2/CCR2通过Cx43及ERK1/2/MAPK信号通路调控NMDA活性等。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。