PKMζ—Kalirin-7—AMPA受体GluR1膜上位在瑞芬太尼二次痛敏中的调控机制

Remifentanil-induced hyperalgesia is followed by hyperalgesic memory, a second surgery with remifentanil anesthesia is identified to generate greater secondary postoperative hyperalgesia, but its mechanism remains elusive. Our previous studies have manifested that NMDA and AMPA receptors are involved in development and maintenance of remifentanil induced hyperalgesia, moreover, GluR1 membrane insertion is associated with remifentanil-induced hyperalgesia. Recent studies have reported that protein kinase M zeta (PKMζ) mediated AMPA receptor activation is critical for formation of spinal cord memory function— LTP. We also have found that PKMζ could modulate Kalirin-7 expression in remifentanil-induced secondary hyperalgesia in rats. We hypothesize that PKMζ may exert a pivotal effect on remifentanil-induced secondary hyperalgesia. Therefore in this study, following accomplishment of a rat model of remifentanil-induced secondary hyperalgesia, we use nociceptive behavior test, Western blot, co-immunoprecipitation, immunofluorescence and Golgi staining to detect dynamic changes of PKMζ, Kalirin-7, GluR1 and dendritic spines structure in remifentanil-induced secondary hyperalgesia. We also culture rat primary spinal dorsal horn neurons and spinal cord slice, and inhibit PKMζ and Kalirin-7 to demonstrate the role of PKMζ— Kalirin-7—GluR1 membrane insertion in remifentanil-induced secondary hyperalgesia. The present study will provide the novel options for the effective prevention of secondary hyperalgesia induced by remifentanil in clinics.

瑞芬太尼痛敏后可形成痛敏记忆，二次手术再次使用瑞芬太尼会引发更严重的二次痛敏，其机制尚不明确。课题组已发表工作证实NMDA和AMPA受体均参与瑞芬太尼痛敏形成和维持, 其中AMPA受体GluR1膜上位与瑞芬太尼痛敏密切相关。新近文献报道PKMζ调控AMPA受体活化可维持脊髓记忆功能LTP产生，课题组也发现PKMζ可调节瑞芬太尼二次痛敏大鼠Kalirin-7表达，故推测PKMζ是介导瑞芬太尼二次痛敏的关键。本研究拟建立大鼠在体瑞芬太尼二次痛敏模型，利用痛敏行为学测定、Western blot、共沉淀、荧光和高尔基染色研究PKMζ、Kalirin-7、GluR1和树突棘形态在瑞芬太尼二次痛敏中动态变化；体外培养大鼠脊髓片和背角神经元，抑制PKMζ和Kalirin-7研究PKMζ—Kalirin-7—GluR1膜上位在瑞芬太尼二次痛敏中的机制。本研究将为瑞芬太尼二次痛敏的临床防治提供新思路。

瑞芬太尼是超短效μ-阿片受体激动剂，广泛用于外科手术全身麻醉的镇痛，其引发的痛觉过敏（OIH）现象发生普遍。二次手术临床常见,而二次手术全身麻醉再次使用瑞芬太尼镇痛是否形成痛敏记忆并引发更严重的二次痛敏尚无定论。新近文献报道PKMζ调控AMPA受体活化可维持脊髓记忆功能LTP产生，课题组也发现PKMζ可调节瑞芬太尼二次痛敏大鼠Kalirin-7表达，故推测PKMζ是介导瑞芬太尼二次痛敏的关键。本研究制作大鼠在体瑞芬太尼二次痛敏模型，利用痛敏行为学测定、Western blot、免疫共沉淀、荧光和高尔基染色，完成了PKMζ、Kalirin-7、GluR1和树突棘形态在瑞芬太尼二次痛敏中动态变化的检测；并体外培养大鼠脊髓片和背角神经元，利用PKMζ和Kalirin-7小干扰RNA以及全细胞膜片钳技术研究PKMζ—Kalirin-7—GluR1膜上位在瑞芬太尼二次痛敏中的调控作用。研究发现瑞芬太尼诱发二次痛敏较一次痛敏发生时间早，程度加重，且持续时间延长；二次应用瑞芬太尼后，大鼠脊髓背角中PKMζ和Kalirin-7表达增高，活性增强；脊髓总蛋白、膜蛋白中GluR1亚基表达增高，同时GluR1膜上位上调；二次痛敏大鼠脊髓背角树突棘增大，成熟树突棘数量增加，功能性突触形成；应用PKMζ选择性抑制剂ZIP，Kalirin-7慢病毒转染及GluR1反义寡核苷酸可有效抑制脊髓背角神经元树突棘结构重塑，降低瑞芬太尼诱发二次痛敏程度，缩短二次痛敏持续时间；瑞芬太尼二次孵育后可增加脊髓背角神经元AMPA受体介导的mEPSC的振幅和频率，诱发C纤维LTP形成，而抑制PKMζ—Kalirin-7—GluR1可有效削弱这一效应。本项目在国内外发表论文24篇, 其中SCI 12篇。本研究以AMPA受体GluR1亚单位作为切入点，从整体水平出发，通过在体和离体实验，系统探讨并阐明了PKMζ—Kalirin-7—GluR1信号通路在瑞芬太尼引发二次痛觉过敏形成中的调控机制，为临床合理有效地防治瑞芬太尼引发二次痛敏提供新的靶点。