穴位“Piezos－ATP－腺苷－A1受体”信号轴在针刺镇痛中的机制研究

Needling acupuncture is a classical therapy in traditional Chinese medicine, which stimulates the acupoints with an original and simple method, mechanical force. Cellular ATP release is a common response to mechanical stimulation. The secreted ATP is further indirectly hydrolyzed into adenosine that plays analgesia effects by binding A1 receptors (A1R). Piezo channels are recently discovered mechanical sensitive ion channels. They wildly distribute in skin, neurons and various organs. .This project plans to research the relationship between Piezo channels and ATP release, and the contribution of “Piezo2-ATP-adenosine-A1R” signal axis in acupoints to the analgesia mechanisms in acupuncture. .In vivo tests, acute inflammatory pain will be established on left joints of rats. The thermal pain-relief by needling acupuncture on Housanli acupoints (ST 36) will be assessed. Transient knockdown of Piezos in ST 36 with RNA interference methods will be applied to judge the role of Piezos in analgesia mechanisms of acupuncture. The involvement of adenosine or A1R in this process will be detected by blockage of adenosine generation or inhibition of A1R by inhibitors injection into St 36. .The peripheral endings of dorsal root ganglion (DRG) neurons distributed humane boding including acupoints. In vitro studies, the real-time ATP luminescence imaging and sniffer patch technologies will be applied to analyze the contents of ATP and adenosine in outside space of rats DRG neurons. Finally, in co-existing model, the response of intracellular calcium and A1R currents of DRG neurons to surrounding force activating-epithelium cell will by assessed fluorescence and patch clamp methods. .These investigations explore the transduction and transference of mechanical force to biological analgesia signals in acupoints. The results will contribute to the deeper understanding of the underlying peripheral mechanisms of acupuncture analgesia.

与全身性用药相比,局部治疗是缓解疼痛更明智更安全的手段。针刺无疑具有局部镇痛效应。但其中机制还有待深入探索。.手针是利用原始而简单的机械力施加于穴位以发挥复杂生物效应的治疗手段。本项目从Piezos通道（一种新型的高机械敏感性离子通道）为重点和出发点，以其下游ATP释放、水解及腺苷生成为中间环节，以疼痛相关A1受体为最终目标，研究外周神经“Piezos－ATP－腺苷－A1受体”信号轴在针刺局部镇痛机制中的作用。.首先，以急性踝关节炎大鼠为模型，针刺后三里穴，以痛阈为主要检测指标，结合基因干扰等技术，分析该信号轴在针刺镇痛机制中的作用；此外，结合穴位特异性，以大鼠DRG神经元为对象，利用生物萤光成像、Sniffer patch和钙荧光技术，研究该信号轴在神经元本身和神经元对周围细胞的信息应答中的作用。.本项目在外周神经上，探究机械力信号和生物疼痛信号间的相互作用，以深入了解针灸的局部镇痛机制

针刺是一种安全、有效的镇痛疗法。它利用原始而简单的机械力施加于穴位以发挥复杂生物效应。本项目从机械敏感性Piezos通道出发，以穴区ATP等嘌呤物质为对象，从穴区神经和非神经组织中研究针刺镇痛（Acupuncture analgesia, AA）的机制。.动物实验中，在清醒无束缚大鼠上证明单次20min针刺ST36能有效缓解踝关节炎导致的机械痛和热痛（P均<0.001，vs.模型组）。穴位机械敏感性蛋白抑制（钌红或钆注射）能明显抑制AA疗效（P均<0.05，vs.非注射）。Piezos蛋白特异性抑制（Gsmtx4注射）有相同的疗效（P<0.001，vs.非注射）。利用生物素酶法和高效液相法对穴区微透析液分析可知，针刺可导致ATP和腺苷一过性上升。抑制穴区ATP水解（ARL67156注射）可明显降低AA疗效（P<0.01，vs.非注射）。激活穴区腺苷A1受体（CCPA注射）可模拟AA作用（P<0.05，vs.模型组）。RT-PCR和免疫荧光检测均证明穴区表达机械敏感性Piezo1和Piezo2蛋白。.细胞实验中，免疫荧光标记显示大鼠DRG神经元表达piezo2蛋白。机械刺激（MS）可导致36% DRG神经元出现[Ca2+]i上升。其中适应性（adapted）[Ca2+]i反应可被钌红抑制。此外，MS可促进大鼠腰段L3-5 DRG释放ATP增加（P<0.001）。并且这种MS诱导的ATP释放在模型组明显增多（P<0.001，vs. 空白组），在针刺组又被显著下调（P<0.001，vs. 模型组）。在非神经元，以人HMC-1细胞和大鼠RBL-2H3细胞为对象，MS可导致两者释放ATP增多（P均<0.05）。在HMC-1上，MS诱导的ATP释放依赖于[Ca2+]i上升。在RBL-2H3上发现，MS诱导ATP释放机制涉及P2Y13，P2X7和A1受体。在DRG和RBL-2H3细胞共存的环境中，DRG的存在可明显水解MS诱导的ATP释放（P<0.001）。RT-PCR结果证明，大鼠DRG和坐骨神经干上表达具有水解ATP能力的多种核苷酸水解酶mRNA。.综上所述，穴区Piezo通道参与手针AA机制，穴区ATP和Ado水平的增加，A1受体的激活可能是其下游通路。穴位神经和非神经组织的机械敏感性相关的嘌呤能信号均可能参与其中。该研究为AA的穴位机制提供客观的实验室依据。