基于TLR2信号通路的“针刺止痒”中枢神经传导机制研究

Pruritus is a common clinical symptom. Severe pruritus may cause malaise no less than chronic pain. Acupuncture treatment for pruritus has good effect, but the mechanism of acupuncture anti-pruritic effect is still poorly understood in central nervous conduction. In our previous study, we found that TLR2 might participate in the central nervous conduction of pruritus, and some microRNA might be important regulatory factors in the signal pathway of TLR2. Acupuncture could adjust TRL2 expression in central level according to literatures. Thus we speculate the anti-pruritic effect of acupuncture has close relationship with TLR2. We apply acupuncture preconditioning in the acupoints of LI11 and SP10. With wild type and TLR2 gene knockout mice models of morphine induced pruritus taken as a research pointcut, peripheral pruritic cause will be eliminated and nerve condution of pain will be suppressed. TRL2 signal pathway will be upregulated and downregulated in order to confirm TLR2 functions in acupuncture anti-pruritic effect in central nervous conduction. We will also study the nerve impulses in the level of spinal cord dorsal horn neurons and its related neurotransmitters. Furthermore, microRNA chip screening will be applied to determine the differential microRNA in cerebrospinal fluid and spinal cord dorsal horn neurons, and target gene test will be completed. The project will be expected to illuminate the regulating mechanism of TLR2 signal pathway in acupuncture anti-pruritic effect in the process of nervous conduction, and to preliminarily explore the mechanism of acupuncture part-whole effect in central nervous conduction.

瘙痒是常见的临床症状，严重的瘙痒给病患造成的痛苦程度并不亚于慢性疼痛。针刺治疗瘙痒具有良好疗效，但对于针刺止痒效应的中枢神经传导机制仍知之甚少。我们在前期研究中发现TLR2可能参与瘙痒的中枢神经传导，某些microRNA可能是TLR2信号通路的重要调控因子，而针刺可调节TLR2中枢水平表达，由此我们推测针刺止痒效应与TLR2有密切关系。我们采用针刺预处理曲池、血海，以吗啡诱导的野生型和TLR2基因敲除小鼠瘙痒模型作为研究切入点，在排除外周瘙痒病因作用和抑制疼痛神经传导情况下, 上调和下调TLR2信号通路，证实TLR2在针刺止痒中枢神经传导的作用；同时研究脊髓背角神经元水平的神经冲动及相关神经递质，使用microRNA芯片筛选，确定脑脊液和脊髓背角神经元中的差异microRNA并完成靶基因测试，有望明确针刺止痒神经传导过程中TLR2信号通路的调控机制，初步探索针刺整体效应的中枢神经传导机制。

针灸治疗瘙痒具有良好疗效，但对其效应机制知之甚少。瘙痒与疼痛有不同的神经发生和传导机制，因此“针刺止痒”应有别于“针刺止痛”的中枢神经传导机制。本研究使用BALB/c背景的野生型小鼠及TLR2基因敲除小鼠，建立吗啡鞘内注射诱导中枢性瘙痒小鼠模型，针刺预处理（逆针灸）选穴曲池、血海。主要研究内容为：通过TLR2基因敲除和TLR2配体（PGN）激活，下调和上调TLR2信号通路，观察小鼠瘙痒行为学的差异，检测脊髓水平TLR2以及下游MyD88、TRAF6、NF-κB表达的变化、炎症因子的表达水平和免疫荧光双染直接观察小鼠脊髓神经元膜和树突的变化，比较空白组、模型组、电针组和电针对照组，验证TLR2信号通路在脊髓水平与“针刺止痒”效应的关系。通过在体膜片钳技术，研究“针刺止痒”效应在脊髓背角神经元水平的神经电生理变化。行为学检查发现，针刺曲池、血海或下调TLR2信号通路均可明显减轻瘙痒。RT-PCR、mRNA 和western blot检测证实，针刺减弱TLR2 、MyD88、 TRAF6和NF-κB的表达。免疫荧光双染与上述结果相吻合。针刺可通过减少促炎相关因子IL-1、IL-6和TNF-α的表达，升高抑炎相关因子IL-10的表达而减轻相关炎症反应。针刺可下调M1-和上调M2-调控巨噬细胞分化。在体膜片钳检查发现神经元之间的神经网络能够接收突触前的信号输入。由此得出以下结论：针刺曲池、血海对中枢性瘙痒具有止痒效应，且具有腧穴相对特异性；TLR2信号通路参与瘙痒中枢神经传导；针刺通过下调脊髓水平TLR2-MyD88-TRAF6-NF-κB通路发挥止痒作用。该课题结果为下一步开展防治吗啡鞘注诱发瘙痒不良反应的针灸临床研究提供确切、充分的实验依据；为针灸治疗瘙痒性皮肤病经穴特异性选择提供实验依据；为探索脊髓水平针刺整体效应的中枢神经传导机制创造条件；通过抑制或下调TLR2信号通路可发挥止痒作用，为探寻治疗瘙痒的新方法和新药物提供线索，具有重大的科研和现实意义。