脊髓水平Apelin/APJ在慢性炎症痛中的作用和机制研究

The neuropeptide apelin is the endogenous ligand for APJ, a G protein-coupled receptor (GPCR). Apelin/APJ system is widely distributed in central nervous system and peripheral tissues, and involved in a broad range of physiological and pathophysiological processes. Our previous reports demonstrate that apelin-13 has played a role in regulating acetic acid-induced visceral pain and acute pain (tail-flick test) at spinal level in mice. Our preliminary experiment shows that intrathecal (i.t.) administration of apelin-13 induced hyperalgesia in the chronic inflammatory pain mouse model. However, its mechanism is still unknown. The present proposal is designed to synthesize apelin using solid-phase synthesis method and establish a mouse model of chronic inflammatory pain induced by complete Freund's adjuvant (CFA). Mice will be i.t. injected with apelin and antagonists/inhibitors, and the pain-related behavior will be evaluated to study the regulatory effect of apelin on chronic inflammatory pain. Whether the effect of apelin/APJ on NMDA receptor-mediated synaptic transmission is through the PKC/Ras/Raf/MEK/ERK1/2 signal transduction pathway in the spinal dorsal horn will be investigated, using methods such as dual-immunofluorescence, confocal laser scanning microscope (CLSM), patch-clamp recording technique, western blot, etc. The molecular mechanism of apelin/APJ on the chronic inflammatory pain will be indicated. The results could provide theoretical basis for the clinical application of apelin/APJ system, a potential analgesic drug target.

神经肽Apelin是G蛋白偶联受体APJ的内源性配体，广泛分布于中枢神经系统和外周组织，参与多种生理和病理生理过程的调节。前期工作发现脊髓水平Apelin-13在醋酸引起的内脏痛和热甩尾急性痛模型中发挥调节作用。预实验表明鞘内注射Apelin-13在慢性炎症痛模型中引起痛敏反应，但其作用机制不清楚。本项目拟用固相法合成Apelin，建立完全弗氏佐剂诱导的小鼠慢性炎症痛模型，鞘内注射Apelin及拮抗剂/抑制剂，通过痛觉行为学测试探究Apelin对慢性炎症痛的调节作用；同时用免疫荧光双标记、激光共聚焦、膜片钳、Western印迹等方法研究Apelin/APJ在脊髓背角是否通过PKC/Ras/Raf/MEK/ERK1/2信号转导通路调节NMDA受体介导的痛觉突触传递，揭示Apelin/APJ对慢性炎症痛调节的分子机制。研究结果可为潜在镇痛药物靶点Apelin/APJ系统的临床应用提供理论依据。

神经肽Apelin作为G蛋白偶联受体APJ的内源性配体，具有多种生物学效应，但Apelin/APJ系统在炎症痛中的作用尚不明确。课题组用完全弗氏佐剂（CFA）诱导的慢性炎症痛模型和福尔马林诱导的急性炎症痛模型探究Apelin/APJ对痛觉的调节作用及机制。结果发现CFA诱导小鼠脊髓L4/5节段Apln mRNA下调；鞘内注射apelin-13（1-10 nmol）明显抑制由温度刺激和机械刺激引起的CFA小鼠痛敏反应，该作用可被APJ拮抗剂所阻断；apelin-13的镇痛效果比apelin-36和(pyr)apelin-13强。鞘内注射apelin-13能够明显逆转CFA诱导的小鼠脊髓L4/5节段Aplnr mRNA水平的下降，Grin2b、Camk2d 和c-Fos mRNA水平的升高，谷氨酸含量和NR2B蛋白表达的升高。鞘内注射apelin-13明显降低CFA小鼠髓背角Ⅳ+Ⅴ层c-Fos免疫阳性神经元数目。这些结果表明apelin-13抑制髓背角Ⅳ+Ⅴ层神经元活动，APJ/CaMK2/NR2B参与了apelin-13对慢性炎症痛小鼠的镇痛效应。此外，静脉注射 (pyr)apelin-13（10-20 mg/kg）在福尔马林致痛小鼠第二时相有明显镇痛作用；该作用可被apelin-13(F13A)、naloxone和nor-BNI所逆转；(pyr)apelin-13明显提高小鼠前额皮层Dynorphin和KOR的基因和蛋白表达，提示(pyr)apelin-13对急性炎症痛小鼠的镇痛作用是通过Dynorphin/KOR系统起作用的。另外，我们还发现侧脑室注射神经肽spexin（2.43 mg/kg）在福尔马林致痛模型小鼠第一和第二时相均有显著镇痛作用，基因芯片分析结果显示脑组织Fos基因表达量增加量2.3倍，免疫组化结果表明spexin显著增加导水管周围灰质尾部背内侧区、外侧区和腹外侧区c-Fos免疫阳性细胞数，说明中枢spexin通过Fos抑制小鼠急性炎症痛。总之，Apelin在慢性炎症痛及急性炎症痛中均发挥镇痛作用，该作用通过NMDA受体2和kappa阿片受体介导。研究结果可为Apelin/APJ系统作为新的潜在镇痛药物靶点提供实验基础和理论依据，对炎症痛的发病机理阐明和治疗具有指导意义。