抑制脊髓AMPK活性降低神经元游离钙离子减缓神经病理疼痛

The phosphorylation of receptors and ion channels in the spinal sensory nervous circuit, is the key of peripheral and central sensory nervous circuit sensitization. It is well known that intraperitoneal injection of AICAR can improve pain threshold of mice undergone CCI by phosphorylation of AMPK, however, the applicant found that intrathecal injection of AICAR could not increase the pain threshold of mice, but decrease it. So we speculated that the different pharmacological effect may be due to different drug delivery route. Intraperitoneal injection of AICAR reduce the release of inflammatory cytokines, while intrathecal injection of AICAR excites neurons. Based on the studies of AMPK, we speculated that the phosphorylation of AMPK could activate transcription factor, P53, subsequently promote its downstream gene NNAT expression via lincRNA-p21, and promote endoplasmic reticulum Ca2+ release by NNAT located in the endoplasmic reticulum. Our preliminary experiment showed that intrathecal injection of small interfering RNA of AMPK, Trp53, NNAT or compound C also increase the pain threshod of mice with CCI or induce-pain by AICAR, and provided evidence for this speculation. Clarification of signal transmission and its functions of regulating mechanism underlying the regulation of AMPK in NNAT, which provide breakthrough of theory in neuroscience related to pain, provides a new idea for the treatment of drugs, and a novel target.

脊髓感觉神经环路中的受体和离子通道磷酸化，是外周和中枢神经环路的感觉敏感化的关键。已有研究表明腹腔注射AICAR使AMPK磷酸化可提高CCI小鼠痛阈，而申请者研究发现鞘内注射AICAR不能提高疼阈，却降低正常小鼠痛阈。推测可能是不同给药途径药理作用不同，腹腔注射AICAR可减少炎性因子释放，而鞘内注射则引起神经元兴奋。综合AMPK的相关文献，我们推测，磷酸化的AMPK可活化转录因子P53，然后通过lincRNA-p21促进其下游基因NNAT表达，定位于内质网上的NNAT促进内质网Ca2+释放。本课题组前期结果表明，CCI病理性慢性疼痛小鼠和AICAR致痛小鼠，鞘内注射小干扰AMPK、Trp53、NNAT或复合物C都可提高痛阈，为以上推测提供了重要证据。阐明AMPK对NNAT调控导致的信号传递及其功能的调节机制，势必为相关疼痛神经科学理论的突破和理想治疗药物的研发提供新的思路和有益的靶标。

研究背景：脊髓感觉神经环路中的受体和离子通道磷酸化，是外周和中枢神经环路的感觉敏感化的关键。已有研究表明腹腔注射AICAR使AMPK磷酸化可提高CCI小鼠痛阈，而申请者研究发现正常小鼠鞘内注射AICAR不能提高疼阈，却降低正常小鼠痛阈。.研究内容：1 研究AMPK 与CCI 神经病理性慢性疼痛的关系；. 2研究pAMPK 调控P53 进而影响疼痛的分子机制。.研究结果：AICAR引起的正常小鼠痛觉过敏以及CCI小鼠的痛觉过敏可被鞘内注射siP53逆转。CCI小鼠鞘内注射siAMPK表达可起镇痛作用，在CCI小鼠鞘内注射si AMPKα2起镇痛作用，而鞘内注射si AMPKα1镇痛作用不明显。在CCI小鼠，过表达AMPKα2可使DUSP1磷酸化表达减少，从而诱导 p38/JNK 的活化，导致p-P38、JNK、ERK表达增加。.科学意义：本研究在组织水平、细胞水平研究 AMPK 调控p53表达介导的痛觉过敏机制。在中枢神经系统中发现 AMPK调控p53作用的新机制，将为AMPK、P53与慢性疼痛的发生、发展的关系提供直接的科学证据。因此 本项目的意义在于:能丰富临床医学、分子生物学、病理学等学科的相关基础理论，揭示 AMPK 以及P53调节慢性疼痛的分子机制，为临床上研发理想的治疗慢性疼痛药物提供新的靶标。