Signaling Mechanisms of Opioid-Induced Hyperalgesia and Tolerance

阿片类药物诱发痛敏和耐受的信号转导机制 项目摘要 该项目的主要目标是确定负责开发的关键信号机制， 阿片样物质诱导的痛觉过敏和镇痛耐受（OHT）。阿片类药物仍然是治疗不可缺少的 由手术、创伤和癌症引起的剧烈疼痛。然而，急性和重复给予μ-阿片样物质， 受体（莫尔）激动剂经常引起OHT，这是阿片类药物充分缓解疼痛的主要障碍。OHT可以 也会导致不安全的阿片类药物剂量增加，导致依赖、成瘾，甚至过量死亡。阿片 信号传导是复杂的，并且已经主要在体外进行了研究，但是各种信号传导的功能意义和相关性是不确定的。 OHT的阿片样物质信号传导组分知之甚少。N-甲基-D-天冬氨酸受体（NMDAR）是一种 临床验证的治疗OHT的靶标，并且细胞外信号调节激酶（ERK）被莫尔刺激 在脊髓水平和OHT中参与阿片样物质诱导的NMDAR过度活跃。目前， 对于脊髓水平上导致ERK刺激的上游信号传导机制知之甚少 在OHT期间。虽然BRAF是一种丝氨酸/苏氨酸特异性蛋白激酶，是ERK的重要上游信号， 激活，其在OHT中的作用以前没有被认识到。在我们的初步研究中，我们发现， 用阿片类药物治疗增加了脊髓中的BRAF活性。此外，BRAF抑制或敲低 在脊髓水平上显著减弱了OHT，挽救了突触运输和表达， 阿片类药物治疗改变了脊髓中的MORs和NMDAR。这些初步研究结果表明，BRAF- 在发育过程中，依赖性信号在控制突触莫尔和NMDAR可塑性中起关键作用 的OHT。因此，在这个竞争性的更新应用程序中，我们将测试重复的总体假设， 通过BRAF介导的信号传导轴，用阿片类药物治疗诱导（1）通过抑制 初级传入中枢终末MORs的表达和活性;（2）促进痛觉过敏 NMDAR在与脊髓兴奋性神经元突触的初级传入终末的运输和活性。 为了验证这一假设，我们将使用多学科的方法，包括蛋白质生物化学， 脊髓切片中的电生理记录，以及靶向基因敲除和敲入。我们提出的 这些研究有望促进我们对与以下高度相关的基本信号机制的理解： OHT的发展。我们的项目也有重要的临床意义，并可能导致新的战略 (e.g.,使用FDA批准的BRAF抑制剂）用于改善患有严重疼痛的患者中的阿片类镇痛功效。

Signaling Mechanisms of Opioid-induced Hyperalgesia and Tolerance Project Summary The major objective of this project is to identify key signaling mechanisms responsible for the development of opioid-induced hyperalgesia and analgesic tolerance (OHT). Opioid drugs remain indispensable for treating severe pain caused by surgery, trauma, and cancer. However, acute and repeated administration of μ-opioid receptor (MOR) agonists often cause OHT, the major obstacle to adequate pain relief with opioids. OHT can also lead to unsafe opioid dose escalation, resulting in dependence, addiction, and even overdose death. Opioid signaling is complex and has been studied mostly in vitro, but the functional significance and relevance of various opioid signaling components to OHT are poorly understood. N-methyl-D-aspartate receptors (NMDARs) are a clinically validated target for treating OHT, and extracellular signal-regulated kinase (ERK) is stimulated by MOR activation and is involved in opioid-induced NMDAR hyperactivity at the spinal cord level and in OHT. At present, little is known about the upstream signaling mechanism leading to stimulation of ERK at the spinal cord level during OHT. Although BRAF, a serine/threonine-specific protein kinase, is a crucial upstream signal for ERK activation, its role in OHT has not been recognized previously. In our preliminary studies, we found that repeated treatment with opioids increased BRAF activity in the spinal cord. Furthermore, BRAF inhibition or knockdown at the spinal cord level substantially attenuated OHT and rescued the synaptic trafficking and expression of MORs and NMDARs in the spinal cord altered by opioid treatment. These initial findings suggest that BRAF- dependent signaling plays a key role in the control of synaptic MOR and NMDAR plasticity in the development of OHT. Therefore, in this competing renewal application, we will test the overall hypothesis that repeated treatment with opioids, through the BRAF-mediated signaling axis, induces (1) analgesic tolerance by inhibiting expression and activity of MORs at primary afferent central terminals and (2) hyperalgesia by promoting trafficking and activity of NMDARs at primary afferent terminals synapsing with spinal cord excitatory neurons. To test this hypothesis, we will use a multidisciplinary approach, including protein biochemistry, electrophysiological recordings in spinal cord slices, and targeted gene knockout and knockin. Our proposed studies are expected to advance our understanding of the fundamental signaling mechanisms highly relevant to the development of OHT. Our project also has important clinical implications and could lead to new strategies (e.g., using FDA-approved BRAF inhibitors) for improving opioid analgesic efficacy in patients with severe pain.