脊髓水平MOR-NR1/NR2B-MOR信号通路在瑞芬太尼诱发的术后痛觉过敏中的作用及机制研究

Remifentanil-indued hyperalgesia is a difficult problem that limits its clinical application. The molecular mechanisms of remifentanil-indued hyperalgesia is still unclear. Our previous studies showed that the phosphorylation of NMDA receptor containing NR2B subunit in spinal dorsal horn plays an important role in the mechanisms of various pain including remifentanil-indued hyperalgesia, bone cancer pain and so on. Recent research indicated that the C-terminus of mu-opioid receptors (MOR) binds directly to NR1 subunit carrying the C1 region. This MOR-NMDAR association participates in the pain signal regulation in the process of morphine tolerance. Morphine disrupts this association by protein kinase-C (PKC)-mediated phosphorylation of the NR1 C1 segment. In our study, we will investigate the dynamic changes of MOR-NMDAR association, and the phosphorylation of MOR, NR1 and NR2B in spinal dorsal horn during the process of remifentanil-induced hyperalgesia in rats. Moreover, we will utilize the selectivity inhibitor of MOR naloxone, the specific inhibitor of NR2B Ifenprodil, the antagonist of glycine CGP61594, the specific inhibitor of PKC Go7874, the specific inhibitor of Src Genistein and the specific inhibitor of CaMKⅡ KN62 to investigate the mechanisms of spinal MOR-NMDAR association and regulation, and MOR-NR1/NR2B-MOR signal pathway in remifentanil-indued hyperalgesia. The double-label immunofluorescence technique, co-immunoprecipitation, western blot and real time PCR will be applied to locate the co-exist and quantify the expression and phosphorylation of MOR, NR1 and NR2B in vivo and in vitro. The implementation of this project provides experimental evidence for the rational use of remifentanil in clinical, and provides novel intervention targets for the prophylaxis and treatment of remifentanil-indued hyperalgesia.

瑞芬太尼致痛觉过敏是限制其临床应用的一大医学难题。我们前期研究发现脊髓背角含NR2B亚单位的NMDAR磷酸化在瑞芬太尼致痛觉过敏等多种疼痛中起重要作用。近期研究表明在中枢神经系统MOR通过C末端与NMDAR的NR1亚基的C1末端直接结合，吗啡通过MOR活化蛋白激酶C引起NR1磷酸化，从而促使MOR磷酸化，两者间的结合解离，MOR内化脱敏，从而在吗啡耐受中发挥重要作用。本项目拟通过评价瑞芬太尼诱发切口痛大鼠痛觉过敏时脊髓背角MOR与NR1的免疫共沉淀变化以及MOR、NR1、NR2B磷酸化水平改变，及特异性MOR拮抗剂、特异性NR2B拮抗剂、甘氨酸拮抗剂及特异性蛋白激酶抑制剂等对其的影响，探讨MOR-NMDAR相互调节，即MOR-NR1/NR2B-MOR信号环路参与瑞芬太尼诱发痛觉过敏的分子机制，为临床合理使用瑞芬太尼和预防其诱发痛觉过敏提供理论依据，为研发双功能靶向镇痛药提供有益靶标。

瑞芬太尼致痛觉过敏是限制其临床应用的一大医学难题。本研究在大鼠瑞芬太尼诱发术后痛觉过敏的模型上，评价脊髓背角MOR、NR1、NR2B磷酸化水平以及MOR与NR1的结合水平，使用相应抑制剂探讨MOR-NMDAR相互调节，即MOR-NR1/NR2B-MOR信号环路参与瑞芬太尼诱发痛觉过敏的机制，为临床合理使用瑞芬太尼和防治其诱发痛觉过敏提供理论依据。我们的研究显示：①在大鼠行切口手术同时，皮下泵注瑞芬太尼可以诱发术后机械痛敏和热痛敏，上调脊髓水平NR1亚基、NR2B亚基与MOR磷酸化，并使NR1与MOR间的结合解离，提示这一过程在瑞芬太尼诱发的痛觉过敏中起关键作用。②鞘内注射甘氨酸拮抗剂CGP61594或NR2B特异性抑制剂艾芬地尔，显著下调脊髓水平NR1、NR2B、MOR磷酸化，增加NR1与MOR的结合水平，缓解瑞芬太尼诱发的术后痛觉过敏。③鞘内注射PKC抑制剂Gö 7874、Src抑制剂Genistein、CaMKII抑制剂KN62，可以分别下调脊髓水平NR1、NR2B、MOR磷酸化水平，增加NR1与MOR的结合率，缓解瑞芬太尼诱发的术后痛觉过敏。④腹腔注射MMP-9抑制剂N-乙酰半胱氨酸可以剂量依赖性的抑制背根节MMP-9的活性，减少IL-1β的剪切与成熟，下调脊髓水平NR1与NR2B磷酸化水平，抑制PKCγ的活性，抑制脊髓水平星形胶质细胞与小胶质细胞的活化，从而缓解瑞芬太尼诱发的术后痛觉过敏。⑤鞘内注射CDK5抑制剂roscovitine可以下调脊髓水平NR2A、NR2B及mGluR5的磷酸化水平，缓解瑞芬太尼诱发的术后痛觉过敏。⑥鞘内注射α7nAchR选择性激动剂PHA-543613和II型正性变构调节剂PNU-120596可呈剂量依赖性地缓解瑞芬太尼诱发痛觉过敏大鼠的机械痛敏和热痛敏，并降低脊髓水平NR2B亚基磷酸化水平以及促炎性细胞因子TNF-α和IL-6的水平。⑦鞘内注射CB2受体激动剂JWH015可以下调脊髓背角NR2B磷酸化，抑制胶质细胞活化及抑制炎症因子，缓解瑞芬太尼诱发的术后痛觉过敏。⑧鞘内注射或脑室内注射NRSF反义寡核苷酸可以解除其对MOR的负性调节，增加MOR的表达，缓解瑞芬太尼诱发的术后痛觉过敏。该课题揭示了脊髓水平MOR-NR1/NR2B-MOR信号环路在瑞芬太尼诱发痛觉过敏中的作用，为其临床防治提供理论依据。