TMEM16C/Slack-GluR1通路在瑞芬太尼诱发痛觉过敏中的调控机制

The incidence of remifentanil-induced hyperalgesia is much higher than other opioids. Until now, there is no effective solution to prevent this phenomenon. The recent studies suggest that TMEM16C/Slack signaling plays a pivotal role in the modulation of pain transmission. Our previous studies suggest that GluR1-containing AMPA receptor plays an important role in remifentanil-induced hyperalgesia via modulate AMPA receptor GluR1 subunit trafficking and receptor function. However, which signaling modulate this phenomenon has not reported yet. This program will use a rat model with incision pain-remifentani-induced hyperalgesia to detect the role of TMEM16C/Slack-CluR1 signaling in remifentanil-induced hyperalgesia. In this study, we use Hot plate test, Von Frey test, Western Blot, Immunofluorescence, Co-IP and Patch-clamp recording to detect the expression and function changes of TMEM16C, Slack and AMPA receptor GluR1 subunit in remifentanil-induced hyperalgesia rats. We also use TMEM16C knockdown and overexpression and GluR1 containing AMPA receptor inhibitor PhTX to demonstrate the role of TMEM16C/Slack—GluR1 signaling in remifentanil-induced hyperalgesia. The current study will provide a new idea for effective prevention and treatment of remifentanil-induced hyperalgesia.

瑞芬太尼引发痛觉过敏临床常见，尚无可靠治疗方法。最新研究显示TMEM16C/Slack通路具有调节痛觉信息传递作用。我们已发表的成果证实含有GluR1亚基AMPA受体表达和功能增加在促进瑞芬太尼痛觉过敏中扮演着重要角色，但含GluR1亚基AMPA受体通过那个通路引起相关作用尚未阐述。本项目将利用切口痛-瑞芬太尼痛觉过敏模型，从在体实验和离体背根神经节培养两个水平，应用热板、Von Frey test、Western Blot、免疫荧光、免疫共沉淀以及膜片钳技术对TMEM16C、Slack以及AMPAR受体GluR1亚基在瑞芬太尼痛觉过敏中的表达和功能的动态变化进行研究，并利用TMEM16C基因沉默和过表达技术，以及含GluR1亚基AMPA受体抑制剂PhTX探讨TMEM16C/Slack—GluR1通路在瑞芬太尼诱发痛觉过敏中的调控机制。本研究将为瑞芬太尼痛觉过敏现象的临床防治提供新思路。

背景 瑞芬太尼是产生痛觉过敏的发生率明显高于其他阿片类药物。TMEM16C / Slack可以负反馈调控AMPA受体介导的内向电流，从而调控神经元兴奋性稳态平衡。我们前期研究发现瑞芬太尼痛觉过敏机制可能与含GluR1亚基AMPA受体的膜蛋白表达和受体电导增加有关。然而, 含GluR1亚基AMPA受体是通过何种信号通路参与瑞芬太尼诱发痛觉过敏的机制尚未深入探索。目前尚无关于TMEM16C/Slack和AMPA受体参与瑞芬太尼痛觉过敏机制的相关研究报道。.主要研究内容 通过在体动物实验，应用大鼠切口痛－瑞芬太尼痛觉过敏模型，明确TMEM16C、Slack及AMPA受体GluR1在瑞芬痛觉过敏中的动态变化及其调控机制。通过离体培养原代脊髓背角神经元实验，应用膜片钳技术，抑制AMPA受体、沉默或过表达TMEM16C，探讨TMEM16C/Slack—GluR1通路在瑞芬太尼诱发痛觉过敏中细胞水平调控机制。.重要结果 正常生理情况下，激活含GluR1亚基AMPA受体可增加离子型通道的电导，增加了Na＋内流，引起Slack通道介导的K＋外向电流，Slack可以负反馈调控AMPA受体介导的内向电流，从而调控神经元兴奋性稳态平衡。瑞芬太尼输注诱发的中枢敏化可以引起AMPA受体GluR1亚基表达增多、TMEM16C和Slack表达下调, 从而抑制了TMEM16C对Slack的调控作用, 降低了Slack电导，从而降低了Slack负反馈调控AMPA受体的作用，增强了脊髓背角的含GluR1亚基AMPA受体电流和动作电位，增加神经元兴奋性，引起瑞芬太尼诱发的痛觉过敏。通过基因沉默TMEM16C发现，单纯的降低TMEM16C就可以增加大鼠机械痛和热痛的阈值，降低Slack蛋白表达，并且增加神经元动作电位和AMPA受体mEPSC，与瑞芬太尼痛觉过敏现象类似。而过表达TMEM16C可增加瑞芬太尼导致的Slack 蛋白表达和电导降低，降低痛觉阈值、神经元动作电位和AMPA受体mEPSC。.科学意义 研究发现瑞芬太尼的痛觉过敏可能是通过TMEM16C/Slack—GluR1通路产生痛觉过敏现象，由于给予AMPA受体抑制剂可影响患者的正常神经系统功能，而给予TMEM16C过表达或Slack受体激动剂则能预防和逆转瑞芬太尼诱发的痛觉过敏的发生，且不影响正常的AMPA受体的生理功能。这为临床合理有效