海马降钙素原在难治性癫痫持续状态发生中的作用机制研究

Refractory Status Epilepticus (RSE) is an intractable neurological emergency, with high lethality and disability rate. The previous studies suggest that inflammation plays a critical role on the initiation and maintenance of status epilepticus (SE), which means that there is an vicious cycle, inflammation→SE→inflammation→RSE. However, the key regulatory molecules involved in the vicious cycle remain unclear. Recently, we found that PCT levels in SE rats model were significantly elevated at the very early stage (5 min post SE), especially in the hippocampus. The increased level of PCT was remarkably higher than that of CRP, IL1, and etc. In addition, our preliminary in vitro studies showed that PCT can reduce the cell surface expression of GABAA receptors and increase the excitability of CA1 pyramidal neurons. These results suggest that PCT might be a key targeting molecule for RSE. Based on the preliminary work, we will seek to clarify the role of hippocampal PCT in the pathophysiological mechanism of RSE. Firstly, we will examine the spatio-temporal expression profile of PCT in the hippocampus of our established LPS-induced RSE model. Then, we will explore the effects of PCT on the trafficking of neuronal excitatory and inhibitory receptors, the electrophysiological properties, and the inflammatory state of glial cells. We will further identify the interacting receptor of PCT and the signaling pathway. Finally, we will investigate the treatment effects of regulating the expression of PCT in the rat hippocampus on RSE. Our study will provide a new therapeutic target for RSE.

难治性癫痫持续状态(RSE)为神经科急症，致死致残率极高，治疗棘手。既往研究表明炎症在癫痫持续状态(SE)的启动及维持中扮演了重要角色，即存在炎症→SE→炎症→RSE这一恶性循环，但在这恶性循环中扮演关键角色的调控分子并不清楚。我课题组最新实验发现在大鼠SE模型早期(5分钟)降钙素原(PCT)大量产生，增高幅度明显强于CRP与IL1等，尤以海马部位表现突出，且PCT可以明显提高神经元兴奋性并下调膜表面抑制性受体的表达，提示：PCT可能是RSE发生中的关键靶点分子。本项目拟在前期研究基础上，采用我们建立的脂多糖诱导RSE动物模型，首先观察该模型海马PCT的时空表达特征；进而探索PCT对神经元兴奋性与抑制性受体膜转运及电生理特性的影响，对胶质细胞炎性状态的作用；进一步鉴定PCT可能的受体及下游信号通路；最后调控海马PCT的表达，观察对RSE的治疗效果。本项目的完成将为RSE的治疗提供新的靶点。

难治性癫痫持续状态(Refractory Status Epileptucus, RSE)为神经科棘手的急危重症，由癫痫持续状态(Status Epileptucus, SE)进展而来。由于RSE 患者海马神经元膜表面抑制性与兴奋性受体表达失衡等原因，临床上常用的苯二氮卓类等抗惊厥药物无法有效控制其发作。RSE 的死亡率高达23-61%，另外有30%患者遗留严重的神经功能障碍。降钙素原(Procalcitonin, PCT)为降钙素的前肽，我课题组前期提出理论假设，RSE后海马PCT含量增加，不但加重了炎症反应，而且导致神经元细胞膜兴奋性与抑制性受体表达失衡，进而改变神经网络兴奋性，引起RSE 的发生及维持。为了验证这一假设，我们在RSE动物模型及离体细胞与脑片中进行了一系列实验，主要研究发现如下：1）在RSE模型中，PCT及其氨基端肽NPCT在血清和大脑海马CA3区的表达量显著升高； PCT/NPCT主要表达于神经元和星形胶质细胞，而在RSE时，升高的PCT/NPCT主要集中于CA3区神经元；2）超微结构及免疫荧光双标染色证实PCT分布于神经元粗面内质网、高尔基体、突触前末梢及突触后结构；3）体外培养原代神经元中发现PCT可使兴奋性谷氨酸受体的NR2B、NR1亚基表达升高，而NPCT不仅可使NR2B、NR1亚基表达升高，还会使抑制性的GABAA受体的GABRG2亚基表达降低；4）PCT与NPCT不引起原代培养神经元胞内cAMP水平升高，提示PCT与NPCT的受体不与cAMP信号偶联；5）PCT对神经元转录本的影响主要富集在免疫炎症反应、而NPCT对神经元转录本的影响主要富集于线粒体氧化呼吸链功能；6）RSE后海马cAMP含量下降，但氧化呼吸链复合体III的活性会反应性升高；7）NPCT会提高原代海马神经元复合体I与复合体IV的活性、提高神经元线粒体膜电位水平；8）PCT会提高海马CA1区锥体神经元兴奋性；9）外周中和PCT会导致RSE大鼠死亡增多，并增加海马退变神经元数量。综上，本项目论证了PCT及NPCT在RSE中的作用及机制，为理解RSE的耐药性提供了新思路，为RSE的治疗提供了新靶点。