NMDA受体NR2B-PN-(-)IRE DMT1通路在瑞芬太尼痛觉过敏中调控机制及H2的治疗作用

Remifentanil-induced hyperalgesia seems to occur more frequently and predictably as it is the ultra short-acting opioid derivative. There is no successful remedy that may prevent this phenomenon. In the previous studies, we found that delta opioid receptor (δ-opioid receptor)-glycogen synthase kinase-3 beta (GSK-3β)-N-methyl-D-aspartate (NMDA) receptor trafficking played a pivotal role in remifentanil-induced hyperalgesia (Nature Science Foundation of China, 30972847, published in 'Anesthesia & Analgesia'). However, which subunit of NMDA receptor and what download pathway of NMDA receptor includued in the mechanisms of remifentanil-induced hyperalgesia are still not clear. Therefore in this study, following accomplishment of a SD rat model of remifentanil-induced hyperalgesia, we use mechanical pain threshold test, thermal pain threshold test, western blotting method, immunofluorescence, enhanced Perl's histochemistry and atomic absorption spectrometry, to detect the changes of NMDA receptor subunit NR2B, peroxynitrite (PN) and (-)IRE divalent metal transporter-1 (DMT1) in remifentanil-induced hyperalgesia. We also culture rat primary spinal dorsal horn neurons and use antagonist of NMDA receptor NR2B, hydrogen-rich culture medium, (-)IRE DMT1 small interfering RNAs (siRNA) to demonstrate the role of NMDA receptor NR2B-PN-(-)IRE DMT1 pathway in remifentanil-induced hyperalgesia. The present study will provide the new method for the effective prevention and treatment of hyperalgesia induced by remifentanil.

瑞芬太尼引发的痛觉过敏临床常见，尚无可靠治疗方法。前期工作证实了δ-阿片受体-GSK3β-NMDA 受体trafficking对瑞芬太尼痛觉过敏的调控机制（国家自然科学基金30972847，发表于Anesthesia & Analgesia），但目前对于NMDA受体何种亚单位及其下游机制在瑞芬太尼痛觉过敏中的作用，尚缺乏报道。本研究制作在体SD 大鼠瑞芬太尼痛觉过敏模型，利用机械性痛阈、热痛敏测定、western blot、免疫荧光、Perl增强染色法和原子吸收分光光度法研究NMDA受体NR2B、PN及(-)IRE DMT1在瑞芬太尼痛觉过敏中动态变化；并培养大鼠背角神经元，利用NR2B拮抗剂、富氢培养基、(-)IRE DMT1 小干扰RNA研究NMDA受体NR2B-PN-(-)IRE DMT1通路在瑞芬太尼痛觉过敏中调控机制。本研究将为瑞芬太尼痛觉过敏现象的临床防治提供新思路。

瑞芬太尼是超短效μ-阿片受体激动剂，广泛用于外科手术全身麻醉的镇痛。但其引发患者术后痛觉过敏(OIH)的发生率明显高于其他阿片类药物。目前对于OIH的发生机制，较肯定的结论是脊髓NMDA受体中枢敏化在其中的关键作用。而瑞芬太尼麻醉后调控何种亚型NMDA受体活化及其下游靶点介导OIH形成尚无定论。本研究制作在体SD大鼠切口痛-瑞芬太尼痛觉过敏模型，利用机械性痛阈、热痛敏测定、western blot、免疫荧光、Perl 增强染色法和原子吸收分光光度法，完成NMDA 受体NR2B、PN 及(–)IRE DMT1 在瑞芬太尼痛觉过敏中动态变化的检测；并培养大鼠背角神经元，利用NR2B 拮抗剂、富氢培养基、(–)IRE DMT1 小干扰RNA以及全细胞膜片钳技术研究NMDA 受体NR2B—PN—(–)IRE DMT1 通路在瑞芬太尼痛觉过敏中调控机制及氢气的治疗作用。研究发现术中1μg•kg-1•min-1瑞芬太尼输注60 min能诱发长时程痛觉过敏；瑞芬太尼痛敏大鼠脊髓背角NR2B膜转位增强，MnSOD 硝化水平升高，且鞘内注射NR2B 选择性拮抗剂能有效增强MnSOD 活性，缓解痛觉过敏；瑞芬太尼痛敏大鼠脊髓背角NT的表达、铁聚集和(-)IRE DMT1的合成明显增多，腹腔注射富氢生理盐水可有效预防瑞芬太尼痛敏，且减少脊髓背角NR2B 亚基表达及膜转位，铁聚集，NT 和(-)IRE DMT1 的表达；(–)IRE DMT1 基因沉默可抑制瑞芬太尼诱发的大鼠脊髓背角神经元内铁离子和钙离子蓄积；瑞芬太尼孵育后可增加脊髓背角神经元NMDA受体介导的mEPSC的振幅和频率，而富氢培养基可有效抑制这一效应。本项目在国内外发表论文30余篇, 其中SCI 14篇。本研究以NMDA受体NR2B亚单位作为切入点，从整体水平出发，通过在体和离体实验，系统探讨并阐明了含NR2B亚基NMDA受体— PN— (-)IRE DMT1信号通路在瑞芬太尼引发的痛觉过敏形成中的调控机制及氢气的保护作用，为今后临床防治OIH提供精准有效的新靶点。