HCN2通道介导LTP参与奥沙利铂诱发神经病理性疼痛的机制研究

The clinical use of oxaliplatin for the treatment of colorectal cancer is limited by the oxaliplatin induced neuropathic pain (OINP). But until now, its pathogenesis remains unclear. Existing research showed that long-term potentiation (LTP) of excitatory postsynaptic potential (EPSP) in spinal dorsal horn is the key procedure of neuropathic pain, underlying an increased efficacy of nociceptive transmission. Besides, recent studies had demonstrated that HCN2 channel plays a crucial role in the synaptic transmission of pain. Our preliminary experiments.indicated that cold hyperalgesia and mechanical allodynia arised in mice receiving oxaliplatin injection, with an enhanced expression of HCN2 channel and elevated frequency of action potential (AP) in dorsal root ganglion (DRG) neurons. We therefore hypothesize that LTP in spinal dorsal horn, induced by the enhanced expression of HCN2 of DRG, critically contributes to the OINP pathogenesis in mice. To verify this hypothesis, we intend to use behavioral tests to validate the effects of oxaliplatin on mice pain thresholds, and then adopt molecular biological techniques to determine HCN2 in DRG and NMDA, pCaMKII in spinal dorsal horn. Patch clamp technique will be used to record AP and LTP in DRG and spinal dorsal horn respectively. At last, gene knockout method, combined with tool medicine, will be employed to validate the relationship between HCN2, AP and LTP and the underlying mechanisms. This work will provide theoretic evidence for.clinical prevention and treatment for OINP.

奥沙利铂用于结直肠癌化疗，诱发的神经性疼痛（OINP）限制了该药物的临床应用，其具体机制尚不清楚。脊髓背角兴奋性突触后电位（EPSP）的长时程增强（LTP），突触传递效能增加，是神经病理性疼痛（NP）的关键环节。最新研究表明HCN2通道在疼痛突触传递中发挥重.要作用。我们前期的研究表明小鼠注射奥沙利铂后出现冷痛觉过敏和触觉痛觉异常，背根神经节（DRG）中HCN2表达增高，神经元放电频率增加。据此我们推测DRG中HCN2上调引起脊髓背角LTP是OINP的重要发病机制。本研究拟利用行为学方法观察奥沙利铂对小鼠痛阈的影响，采用分子生物学技术检测DRG中HCN2和脊髓背角NMDA/pCaMKII表达改变，膜片钳记录DRG神经元动作电位（AP）、脊髓背角LTP，最后通过基因敲除并结合工具药，论证HCN2在OINP中的作用及机制，为临床防治提供理论依据。

我们以往的研究发现，HCN2参与介导了奥沙利铂诱发的神经病理性疼痛。然而，潜在机制仍不完全清楚。在这项研究中，我们发现HFS诱导的长期增强（LTP）后HCN2的水平显着增加，鞘内注射HCN特异性抑制剂ZD7288可逆转腹腔注射奥沙利铂诱发的LTP。此外，我们揭示了奥沙利铂通过激活脊髓背角中的CaMKII / CREB级联反应诱导了脊髓LTP。更重要的是，我们发现奥沙利铂的给药显著增加了兴奋性突触后电流（EPSC）的幅度和动作电位的数量，但被CaMKII抑制剂KN-93和NR2B拮抗剂Ro 25-6981预处理抑制。此外，我们观察到，通过ZD7288预处理，可降低奥沙利铂给药后脊髓N-甲基-d-天冬氨酸（NMDA）受体亚基1（NR1）磷酸化表达的增加。非竞争性NMDA拮抗剂MK-801的给药阻断了奥沙利铂引起的CaMKII / CREB级联激活，阻止了HCN2介导的脊髓LTP诱导和完整大鼠的神经性疼痛行为。所有数据表明，HCN2通过激活NMDA受体介导的CaMKII信号传导诱导脊髓长期增强，从而促进神经性疼痛的发展。本项目综合利用动物行为学、分子生物学、神经电生理等方法深入研究奥沙利铂诱发神经病理性疼痛可能的传导机制，为其防治提供新思路。