Department of Medicine, Mackay Medical College, New Taipei, Taiwan Department of Urology, China Medical University Hospital, China Medical University, Taichung, Taiwan Department of Obstetrics and Gynecology, Chung-Shan Medical University Hospital, Chung-Shan Medical University, Taichung, Taiwan Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan Department of Physiology, College of Medicine, China Medical University, Taichung, Taiwan Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan.
Pain. 2012 Dec;153(12):2380-2392. doi: 10.1016/j.pain.2012.08.004. Epub 2012 Sep 11.
The elusiveness of the mechanism underlying pain is a major impediment in developing effective clinical treatments. We examined whether the phosphorylation of spinal serum- and glucocorticoid-inducible kinase 1 (SGK1) and downstream glutamate receptor interacting protein (GRIP)-associated protein-1 (GRASP-1)/Rab4-dependent GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) recycling play a role in inflammatory pain. After intraplantar injection of complete Freund's adjuvant (CFA), we assessed thermal hyperalgesia using the Hargreaves test and analyzed dorsal horn samples (L4-5) using Western blotting, coprecipitation, and immunofluorescence. CFA administration provoked behavioral hyperalgesia along with SGK1 phosphorylation, GluR1 trafficking from the cytosol to the membrane, and phosphorylated SGK1 (pSGK1)-GRASP-1, GRASP-1-Rab4, and Rab4-GluR1 coprecipitation in the ipsilateral dorsal horn. In the dorsal horns of hyperalgesic rats, CFA-enhanced pSGK1 was demonstrated to be colocalized with NeuN, GRASP-1, Rab4, and GluR1 by immunofluorescence. GSK-650394 (an SGK1 activation antagonist, 1, 10, and 30 μM, 10 μL/rat, intrathecally) dose-dependently prevented CFA-induced pain behavior and the associated SGK1 phosphorylation, GluR1 trafficking, and protein-protein interactions at 1 day after CFA administration. Intrathecal 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an AMPAR antagonist, 1, 3, and 10 μM, 10 μL/rat) attenuated the hyperalgesia and GluR1 trafficking caused by CFA; however, it had no effect on SGK1 phosphorylation. Small interfering RNA targeting Rab4 hindered the CFA-induced hyperalgesia and the associated GluR1 trafficking and Rab4-GluR1 coprecipitation. Our results suggest that spinal SGK1 phosphorylation, which subsequently triggers the GRASP-1/Rab4 cascade, plays a pivotal role in CFA-induced inflammatory pain by regulating GluR1-containing AMPAR recycling in the dorsal horn.
疼痛潜在机制的难以捉摸性是开发有效临床治疗方法的主要障碍。我们研究了脊髓血清和糖皮质激素诱导激酶 1（SGK1）的磷酸化以及下游谷氨酸受体相互作用蛋白（GRIP）相关蛋白-1（GRASP-1）/Rab4 依赖性谷氨酸受体 1 （GluR1）包含的α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（AMPAR）再循环在炎症性疼痛中的作用。在足底注射完全弗氏佐剂（CFA）后，我们使用 Hargreaves 测试评估热痛觉过敏，并使用 Western blot、共沉淀和免疫荧光分析 L4-5 背角样本。CFA 给药引起行为性痛觉过敏，同时伴有 SGK1 磷酸化、GluR1 从细胞质向膜易位以及磷酸化 SGK1（pSGK1）-GRASP-1、GRASP-1-Rab4 和 Rab4-GluR1 在同侧背角的共沉淀。在痛觉过敏大鼠的背角中，免疫荧光显示 CFA 增强的 pSGK1 与 NeuN、GRASP-1、Rab4 和 GluR1 共定位。GSK-650394（一种 SGK1 激活拮抗剂，1、10 和 30 μM，10 μL/大鼠，鞘内）剂量依赖性地预防了 CFA 给药后 1 天引起的疼痛行为以及相关的 SGK1 磷酸化、GluR1 易位和蛋白-蛋白相互作用。鞘内 6-氰基-7-硝基喹喔啉-2,3-二酮（CNQX，一种 AMPAR 拮抗剂，1、3 和 10 μM，10 μL/大鼠）减轻了 CFA 引起的痛觉过敏和 GluR1 易位；然而，它对 SGK1 磷酸化没有影响。靶向 Rab4 的小干扰 RNA 阻碍了 CFA 引起的痛觉过敏和相关的 GluR1 易位和 Rab4-GluR1 共沉淀。我们的结果表明，脊髓 SGK1 磷酸化随后触发 GRASP-1/Rab4 级联反应，通过调节背角中 GluR1 包含的 AMPAR 再循环，在 CFA 诱导的炎症性疼痛中发挥关键作用。
