Signaling Mechanisms of Opioid-Induced Hyperalgesia and Tolerance

阿片类药物痛敏和耐受的信号机制 项目摘要 这个项目的主要目标是确定负责发展的关键信号机制 阿片类药物引起的痛觉过敏和止痛耐受(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，急性和重复使用μ-阿片类药物 受体(MOR)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 还会导致不安全的阿片类药物剂量上升，导致依赖、成瘾，甚至过量死亡。阿片类药物 信号转导是复杂的，而且大多是在体外研究的，但不同的信号转导通路的功能意义和相关性 对OHT的阿片信号成分知之甚少。N-甲基-D-天冬氨酸受体(NMDAR)是一种 临床验证的OHT治疗靶点，MOR刺激细胞外信号调节激酶(ERK) 并参与阿片类药物诱导的脊髓水平和OHT水平的NMDAR过度活动。目前， 目前对导致脊髓水平ERK刺激的上游信号机制知之甚少。 在OHT期间。尽管丝氨酸/苏氨酸特异的蛋白激酶BRAF是ERK的关键上游信号 激活，它在OHT中的作用以前没有被认识到。在我们的初步研究中，我们发现重复 阿片类药物治疗增加了脊髓中BRAF的活性。此外，BRAF抑制或击倒 在脊髓水平显著减弱OHT并挽救突触运输和表达 脊髓中的MORS和NMDAR在阿片类药物治疗后发生改变。这些初步发现表明，BRAF- 依赖信号在突触MOR和NMDAR可塑性发育中起关键作用 OHT的。因此，在这个相互竞争的续订应用程序中，我们将测试重复的总体假设 阿片类药物治疗，通过BRAF介导的信号轴，诱导(1)通过抑制镇痛耐受 MORS在初级传入中枢末梢的表达和活性及(2)促痛敏 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动。 为了验证这一假设，我们将使用多学科方法，包括蛋白质生物化学， 脊髓切片的电生理记录，以及靶向基因敲除和敲击。我们的建议 研究有望促进我们对与以下方面高度相关的基本信号机制的理解 OHT的发展概况。我们的项目还具有重要的临床意义，并可能导致新的策略 (例如，使用FDA批准的BRAF抑制剂)以提高严重疼痛患者的阿片类止痛疗效。

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.