P2Y1-TRPM8-NMDA信号通路在瑞芬太尼诱发痛觉过敏中的作用机制研究

Remifentanil is a kind of narcotic analgesic commonly used in clinical anesthesia. But it can induce hyperalgesia when taking strong analgesic effect, the mechanism of which remains unclear. We have found that the activation of N-methyl-D-aspartate(NMDA) recepter is considered to be an important mechanism of remifentanil-induced hyperalgesia. Inhibition of transient receptor potential melastatin 8(TRPM8) and P2Y1 recepters can both alleviate remifentanil-induced hyperalgesia by downregulating NMDA expression in dorsal root ganglion(DRG), and the inhibition of P2Y1 recepter can also downregulate TRPM8 expression, which implies that the activation of P2Y1-TRPM8-NMDA signal pathway plays an important role in the mechanism of remifentanil-induced hyperalgesia. On the basis of our previous foundings, using RNA interference, behavioral testing, real-time PCR, western blot, immunoflurescence and electrophysiological experiments, this project will attempt to test the alterations of behavioral manifestations and the expression of DRG NMDA in rats with remifentanil-induced hyperalgesia after DRG TRPM8 knockdown by in vivo experiments, and the effect of TRPM8 knockdown on NMDA.function by in vitro experiments, the purpose of which is to clarify the role of TRPM8 in the mechanism of remifentanil-induced hyperalgesia and its regulatory mechanism for NMDA recepter. We will also illustrate the role of P2Y1 in the mechanism of remifentanil-induced hyperalgesia and its regulatory mechanism for TRPM8 recepter using the same methods used to investigate the relationship between TRPM8 and NMDA. The results of this project will finally reveal the concrete role of P2Y1-TRPM8-NMDA signal pathway in the mechanism of remifentanil-induced hyperalgesia.

瑞芬太尼是临床麻醉常用镇痛药，发挥强大镇痛作用的同时也会诱发痛觉过敏，但机制不明。本课题组发现，NMDA激活是瑞芬痛敏重要机制，抑制TRPM8和P2Y1活性都能通过下调背根神经节(DRG)NMDA表达减轻瑞芬痛敏，抑制P2Y1活性还可下调TRPM8表达，提示P2Y1-TRPM8-NMDA通路激活可能在瑞芬痛敏机制中起重要作用。本课题将在前期研究基础上，应用RNA干扰、行为学测试、定量PCR、免疫印迹、免疫荧光和电生理等方法，在体观察TRPM8 knockdown对瑞芬痛敏行为学和DRG NMDA表达影响，离体观察TRPM8 knockdown对NMDA功能影响，阐明TRPM8在瑞芬痛敏机制中的作用及其对NMDA的调控机制，用相同方法阐明P2Y1在瑞芬痛敏机制中的作用及其对TRPM8的调控机制，进而揭示P2Y1-TRPM8-NMDA通路在瑞芬痛敏机制中的具体作用，为防治瑞芬痛敏提供理论依据。

背景.瑞芬太尼具有强大镇痛作用，但其诱发痛觉过敏的比率明显高于其他阿片类药物，目前对其外周机制研究较少。背根神经节NMDA受体的激活可能是瑞芬太尼痛觉过敏的重要机制，TRPM8和P2Y1R对NMDA受体都具有调节作用，但是TRPM8和P2Y1R在瑞芬太尼痛觉过敏中的具体作用及其与NMDA受体的上下游关系尚未见报道。.主要研究内容.利用瑞芬太尼痛觉过敏大鼠模型，研究背根神经节TRPM8膜转运、NMDA受体磷酸化和P2Y1R表达的改变及其在瑞芬太尼痛觉过敏机制中的上下游关系；利用大鼠原代背根神经节神经元，应用膜片钳和钙成像技术测定NMDA受体介导的电流和细胞内钙浓度变化，在离体水平研究P2Y1R-TRPM8-NMDA通路在瑞芬太尼痛觉过敏机制中的作用。.重要结果.瑞芬太尼痛觉过敏大鼠背根神经节TRPM8膜转运和总表达水平、NMDA受体亚型NR1和NR2B的表达和磷酸化及P2Y1R表达水平明显增加，与其机械触诱发痛、热和冷痛觉过敏行为学趋势相符。腹腔注射TRPM8拮抗剂可以削弱瑞芬太尼诱发的冷痛觉过敏并下调背根神经节NR1和NR2B表达和磷酸化，而鞘内注射NMDA受体激动剂会逆转TRPM8拮抗剂的作用。此外，P2Y1R拮抗剂可以明显缓解瑞芬太尼诱发的机械触诱发痛、热和冷痛觉过敏并下调背根神经节NR1和NR2B的表达和磷酸化，其作用也可以被NMDA受体的激活所逆转。进一步研究发现，TRPM8拮抗剂不会影响P2Y1R的表达，但P2Y1R拮抗剂则显著下调TRPM8膜转运水平。离体实验结果显示，背根神经节神经元应用瑞芬太尼孵育后NMDA受体电流及其介导的钙浓度显著增加，TRPM8拮抗剂和P2Y1R拮抗剂都可以显著抑制NMDA受体电流及其介导钙浓度的增加。.科学意义.瑞芬太尼痛觉过敏的中枢机制研究较为广泛，以其为靶点的干预措施可以显著缓解瑞芬太尼诱发的痛觉过敏，但还存在效果有限、副作用明显等问题。本课题在背根神经节这一外周神经系统水平揭示P2Y1R-TRPM8-NMDA信号通路在瑞芬太尼痛觉过敏机制中的作用，为提高治疗效果并减少副作用提供了新的思路。