NR2B-CaMKⅡα-HDAC4 shuttling 信号通路在瑞芬太尼痛觉过敏中的调控机制

Remifentanil-induced hyperalgesia(RIH) is quite common in the clinic, however, there is no effective therapy at present. Our previous studies (published in 《Anesthesiology》and《A&A》) have manifested that NMDA receptor -NR2B subunit trafficking play an important role in the maintenance of remifentanil induced hyperalgesia, but its downstream remains elusive. Recent studies have supported that NR2B subunits are involved in spinal LTP development and maintenance through modulating phosphorylation of CaMKⅡα. HDAC4 shuttling between the nucleus and cytoplasm is critical for synaptic plasticity and pain memory, also regulated by NMDA receptor and CaMKⅡα. Therefore in this study, After performing a rat model of remifentanil-induced hyperalgesia, we use nociceptive behavior test, Western blot, immunofluorescence, immunoelectron microscopy, co-immunoprecipitation, and Golgi staining to detect dynamic changes of NR2B、CaMKⅡα、HDAC4 and dendritic spines structure in remifentanil-induced hyperalgesia. In vitro study, we also culture rat primary spinal dorsal horn neurons, perform RIH model in vitro, and use whole cell patch clamp method to demonstrate the role of NR2B-CaMKⅡα-HDAC4 shuttling in remifentanil-induced hyperalgesia. The present study will provide the novel options for the prevention of remifentanil induced hyperalgesia in clinics.

瑞芬太尼诱发的痛觉过敏（RIH）临床常见，尚无可靠治疗方法。课题组前期工作（发表于Anesthesiology和A&A等）证实NMDA受体NR2B trafficking在RIH中发挥重要调控作用，但其下游机制尚未明确。新近文献证实NR2B可调控CaMKⅡα磷酸化水平，参与脊髓LTP形成与维持。HDAC4胞核和胞浆shuttling在突触可塑性及疼痛记忆中发挥重要作用，且受NMDAR和CaMKⅡα调控。本研究拟建立在体大鼠瑞芬太尼痛觉过敏模型，利用疼痛行为学、western blot、免疫荧光、共沉淀、免疫电镜和高尔基染色等方法探讨NR2B、CaMKⅡα、HDAC4及树突棘形态在RIH中的动态变化；进一步培养大鼠脊髓背角神经元，建立离体RIH模型，利用全细胞膜片钳等技术，研究NR2B-CaMKⅡα-HDAC4 通路在RIH中的调控机制。本研究将为瑞芬太尼痛觉过敏的临床防治提供新思路。

瑞芬太尼引发的痛觉过敏临床常见，尚无可靠治疗方法。课题组前期工作证实了NMDA 受体trafficking对瑞芬太尼痛觉过敏的调控机制，但对于NMDA受体何种亚单位及其下游机制在瑞芬太尼痛觉过敏中的作用，并未做讨论。本研究制作在体SD 大鼠瑞芬太尼痛觉过敏模型，利用机械性痛阈、热痛敏测定、western blot、免疫荧光、免疫电镜等研究NMDA受体NR2B、CaMKⅡα及HDAC4在瑞芬太尼痛觉过敏中动态变化；并培养大鼠脊髓背角神经元，利用NR2B拮抗剂、CaMKⅡα抑制剂，研究NMDA受体NR2B-CaMKⅡα-HDAC4通路在瑞芬太尼痛觉过敏中调控机制。研究结果显示：1.输注瑞芬太尼诱发大鼠机械痛敏和热痛敏，该组脊髓背角pNR2B、pCaMKⅡα、HDAC4表达增加，NR2B/CaMKⅡα二聚体表达增加，脊髓背角神经元胞质中HDAC4表达增加，证实HDAC4发生〈胞浆穿梭改变（shuttling），且脊髓背角神经元树突棘密度显著增加；2. 应用NR2B选择性抑制剂Ro 25-6981、CaMKⅡα选择性抑制剂KN-93后，可以抑制输注瑞芬太尼诱发的大鼠机械痛敏和热痛敏，降低瑞芬太尼引起的pNR2B、pCaMKⅡα、HDAC4及NR2B/CaMKⅡα二聚体表达量的增加，抑制HDAC4 穿梭改变（shuttling），抑制脊髓背角神经元树突棘密度增加。瑞芬太尼可能是通过增加NR2B亚单位膜上表达，进而增强NMDAR介导的钙电流，激活CaMKⅡα，并促使HDAC4磷酸化，并由核内向细胞质中穿梭（shuttling），促使影响突触可塑性的目的基因的转录和表达，进而诱发痛觉过敏。本研究将为瑞芬太尼痛觉过敏现象的临床防治提供新的诊疗思路。