前扣带回KIBRA调节PKMζ/GluR1通路介导开胸术后慢性疼痛的机制研究

We previously demonstrated that chronic post-thoracotomy pain (CPTP) occurs in 30~50% of rats undertaking thoracotomy, and such incidence are significantly increased in rats treated intraoperatively with remifentanil, or postoperatively receiving plantar incision (National Natural Science Foundation of China: 81571071). However, mechanisms underlying the development of CPTP remain unclear. It was reported that the increased activities of PKMζ and its downstream GluR1, one of the AMPA receptor subunits, in the anterior cingulate cortex (ACC) are involved in the maintenance of chronic neuropathic pain. Furthermore, it was demonstrated that KIBRA is associated with the maintenance of memory by inhibiting PKMζ degradation and increasing phosphorylation levels of PKMζ in the hippocampal neurons. Our pilot study found that the protein and mRNA expressions of KIBRA and phosphorylated PKMζ in the ACC were increased in rats with CPTP 14 days after thoracotomy. Therefore, we hypothesize that up-regulation of KIBRA in the ACC causes CPTP through sequentially enhancing the PKMζ activity and GluR1 function. In the present proposal, we will establish that KIBRA and PKMζ in the ACC are implicated in the genesis of CPTP, determine whether PKMζ in the ACC is regulated by KIBRA, and whether synaptic GLuR1 activities in the ACC are regulated by KIBRA and PKMζ. Multidisciplinary approach will be used, including animal behavioral, molecular biology, in vitro electrophysiology assessments, combing with gene therapy using viral vectors injection to induce target genes knockin or knockdown in ACC. Our study will reveal molecular and synaptic mechanisms underlying CPTP, and provide a strategic base for the prevention and treatment of CPTP.

我们发现大鼠开胸术后慢性疼痛发生率约30~50%，术中瑞芬太尼和术后足底切开可增加其发生（国家自然基金：81571071），但是引起该现象的原因不明。有报道前扣带回PKMζ及下游AMPA受体亚基GluR1参与了神经性慢性疼痛维持，海马KIBRA可通过抑制PKMζ降解进而增加磷酸化PKMζ表达参与记忆维持。预实验发现开胸术后有慢性疼痛大鼠在术后14天前扣带回KIBRA及磷酸化PKMζ表达上调。据此我们提出研究假说：开胸手术大鼠前扣带回KIBRA上调可引起PKMζ激活，进而调节GluR1功能介导术后慢性疼痛。本课题拟采用疼痛行为学、分子生物学及体外电生理学等实验方法，结合病毒转染致目的基因在前扣带回过表达或沉默等技术，明确大鼠前扣带回KIBRA和PKMζ参与开胸术后慢性疼痛的形成及KIBRA对PKMζ的调节作用，并探索二者影响GluR1的突触电生理机制，为临床防治术后慢性疼痛提供理论依据。

开胸术后慢性疼痛是直接影响到患者的长期生活和生存质量的严重公共卫生问题。有报道表明前扣带回(ACC)内p-PKMζ参与了神经性慢性疼痛维持，且可能是通过激活AMPA受体亚基GluR1而发挥作用。同时，海马中的KIBRA可通过抑制PKMζ降解进而增加 p-PKMζ的表达参与记忆维持。本研究发现大鼠开胸术后慢性疼痛发生率约30~50%，与临床发生率相一致；开胸术后痛敏阳性大鼠ACC中的p-PKMζ、KIBRA、GluR1均升高，可能参与了开胸术后慢性疼痛的维持；使用PKMζ抑制剂ZIP抑制ACC中p-PKMζ可缓解开胸手术引起的痛觉过敏和胶质细胞激活，同时抑制p-PKMζ、KIBRA、GluR1、TNF-α与IL-1β的过表达；基因沉默ACC中的KIBRA可以降低CPTP发生率，同时抑制p-PKMζ、KIBRA、GluR1、TNF-α与IL-1β的过表达；单纯过表达KIBRA 可以引起大鼠胸壁痛，过表达KIBRA 并行开胸手术可以增加CPTP发生率；KIBRA过表达可增加ACC内KIBRA、p-PKMζ、TNF-α、GluR1、IL-1β的表达，同时可增加CPTP模型大鼠术后21天对侧ACC内KIBRA、p-PKMζ、TNF-α、GluR1、IL-1β的表达；此外，我们发现KIBRA及p-PKMζ参与了开胸术后慢性疼痛大鼠的星型胶质细胞和小胶质细胞显著活化。KIBRA及p-PKMζ是参与大鼠开胸术后慢性疼痛维持的主要分子，二者在介导CPTP中可相互作用，进而引起GluR1、TNF-α、IL-1β的表达变化，同时促进小胶质细胞及星型胶质细胞的活化，参与开胸术后慢性疼痛的维持。p-PKMζ、KIBRA、GluR1、TNF-α与IL-1β均主要存在于神经元，因此推断术后慢性疼痛的维持过程中，神经元及胶质胶质细胞均发挥着主要作用。本研究为临床防治开胸术后慢性疼痛提供理论依据。