脊髓水平MrgC-Gi-NR1/NR2B-nNOS信号通路在骨癌痛中的作用及其机制研究

Treatment of bone cancer pain is a difficult clinical problem. The molecular mechanisms 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 bone cancer pain. Recent research indicated that activated MrgC, which is uniquely distributed in the spinal dorsal root ganglion,can couple with inhibitory protein of Gi. This MrgC-Gi association inhibit the activity of NR1/NR2B by directly or indirectly affecting the phosphorylation. In sequence, the flow of Ca2+ is reduced, the NMDAR- nNOS signaling pathway is weakened, the presynaptic neurotransmitters release into the dorsal horn of the spinal cord and mEPSCs are reduce, which resulted in analgesia.In our study, we will investigate the dynamic changes of MrgC-NMDAR association, and the phosphorylation of MrgC, NR1, NR2B and nNOS in spinal dorsal horn during the process of bone cancer pain in rats. Moreover, we will utilize the agonist of MrgC, the toxins of Gi protein, the antagonists of NR1,the specific inhibitor of NR2B Ifenprodil, the antagonist of glycine CGP61594, the specific inhibitor of nNOS, the specific inhibitor of PKC Go7874 and the specific inhibitor of CaMKⅡ KN62 to investigate the mechanisms of MrgC-Gi-NR1/NR2B-nNOS signaling pathway in bone cancer pian. 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 MrgC, NR1, NR2B and nNOS in vivo and in vitro. The implementation of this project provides experimental evidence for the rational clinical treatment of bone cancer pain, and provides novel intervention targets for the development of specific, efficient, small side effects analgesic drugs.

骨癌痛是临床疼痛治疗的一大医学难题。我们前期研究发现脊髓背角含NR2B亚单位的NMDAR磷酸化在骨癌痛等多种疼痛中起重要作用。近期研究发现独特分布在脊髓背根节的MrgC激活后可与抑制性蛋白Gi耦联，直接或间接影响含NR1/NR2B的NMDAR磷酸化，抑制其活性，使Ca2+内流减少，减弱了NMDAR-nNOS信号通路，减少突触前神经递质释放到脊髓背角，降低mEPSCs，从而产生镇痛作用。本项目拟通过评价骨癌痛时脊髓背角MrgC与NR1的免疫共沉淀变化以及MrgC、NR1、NR2B、nNOS磷酸化水平改变，及MrgC激动剂、Gi毒素、NR1拮抗剂、特异性NR2B拮抗剂、甘氨酸拮抗剂、nNOS拮抗剂及特异性PKC抑制剂等对其的影响，探讨MrgC-Gi-NR1/NR2B-nNOS信号通路参与骨癌痛的调控机制，为临床防治骨癌痛提供理论依据，为研发专一、高效、副作用小的镇痛药物提供新靶点。

骨癌痛是最常见的难治性癌痛之一，对其发病机制及防治的研究至今仍未取得突破。本研究在小鼠骨癌痛模型上，使用MrgC高选择性激动剂、MrgC替代性拮抗剂anti-MrgC抗体、NR2B拮抗剂、nNOS拮抗剂的方法，探讨脊髓水平MrgC-Gαi-NR2B-nNOS信号通路在骨癌痛中的调节作用，为临床控制癌痛提供新的思路和方法。我们的研究结果显示： (1)骨癌痛小鼠脊髓水平MrgC、Gαi、NR2B及nNOS在肿瘤细胞接种后7-14 d时表达显著增加，活性增强，并在时相上与疼痛行为学结果基本一致。(2)骨癌痛小鼠接种后第14 d分别经单次或重复多次鞘内注射不同剂量的MrgC 高选择性激动剂BAM8-22后，中、高剂量组小鼠的NSF显著减少，PWMT显著增高。鞘内给药后1 h疼痛行为学显著改善，2 h达到高峰，12 h后逐渐减弱，24 h后消失，且存在剂量依赖性。而鞘内注射MrgC替代性拮抗剂anti-MrgC抗体则产生了致痛效应。从本实验中可见，BAM8-22可以剂量依赖性的改善骨癌痛。(3) 鞘内注射高剂量BAM8-22能显著上调脊髓水平MrgC、Gαi的表达，下调脊髓水平p-NR2B、t-nNOS及p-nNOS表达，并抑制了脊髓神经元内Ca2+浓度。鞘内注射MrgC替代性拮抗剂anti-MrgC抗体显著下调脊髓水平MrgC、Gαi的表达，同时上调脊髓水平p-NR2B、t-nNOS及p-nNOS表达，且鞘内给药后1 h起效，2 h达到高峰，12 h后逐渐减弱，24 h后作用消失，此与行为学结果基本保持一致。(4) 肿瘤细胞接种后第14 d，干预MrgC-Gαi-NR2B-nNOS信号通路，如采用MrgC高选择性激动剂BAM8-22、MrgC替代性拮抗剂anti-MrgC抗体、NR2B选择性拮抗剂艾芬地尔等，给药后2 h能显著改变骨癌痛小鼠的疼痛行为学，同时调控脊髓水平MrgC、Gαi、NR2B及nNOS的表达及活性。综上提示：激活MrgC后可有效缓解骨癌痛小鼠的疼痛行为学，可能是通过激活Gαi、抑制NR2B/nNOS的表达和活性、抑制脊髓神经元Ca2+内流而发挥作用。该课题揭示脊髓水平MrgC-Gαi-NR2B-nNOS信号通路在骨癌痛形成和发展中的作用，为临床防治癌痛提供理论依据。