中缝核5-HT调控NG2细胞调节睡眠觉醒的作用和机制及酸枣仁汤的干预研究

Insomnia is one of the most common diseases affecting health. Brain 5-HT metabolism disorder is one of the important causes of insomnia. The raphe nuclei, which is rich in 5-HT neurons, is an important neural structure for sleep regulation. NG2 cells are a novel distinct class of central nervous system (CNS) glia cells, and they play critical roles in brain activity. The relationship between the raphe nucleus 5-HT neurons and NG2 glial cells and the roles of NG2 cell in sleep and wakefulness regulation is poorly understood. Preliminary studies have shown that the decline of central 5-HT synthesis is closely related to the activation of astrocytes; effects cortical ATP content and intracellular protein kinase activity, and this effect is related to P2X7 receptor. Research showed that ATP and NG2 cells have close relationship, however whether a decline in 5-HT synthesis induces changes in NG2 cell activity, how NG2 cells communicate with 5-HT systems, and the concomitant effects of such changes are unclear. This experiment examines the relationship between NG2 cells and 5-HT systems in vivo and in vitro, using the immunofluorescence staining, confocal laser scanning microscopy, EEG, microdialysis, high performance liquid chromatography, cell cultures, western blotting and RT-PCR methods. It examines how NG2 cell interacts with 5-HT and activates the subsequent cascades of signal molecules, the roles of NG2 cell in the sleep–wake regulation neural network, and the effects of Suanzaoren decoction on those changes. This study will provide molecular evidence to promote sleep medicine research and development, and to provide new ideas for the prevention and treatment of insomnia.

失眠症是一种影响健康最常见的疾病，脑内5-HT代谢失调是形成失眠的重要原因之一。中缝核内富含5-HT神经元，是调节睡眠和觉醒的重要神经结构， NG2细胞是一种新型的胶质细胞，在中枢神经系统活动中占据非常重要的地位，NG2胶质细胞与5-HT能神经元的关系及在睡眠和觉醒中的作用目前了解甚少。课题组前期实验发现中枢5-HT合成下降与星形胶质细胞激活具有密切关系，影响皮质ATP含量及细胞内蛋白激酶的活性，且该作用与P2X7受体有关。由于ATP及嘌呤受体与NG2细胞有密切功能关系， 5-HT合成下降是否引起NG2细胞活动改变？5-HT系统如何与NG2细胞发生信息交流，并将该作用引向何方？本研究拟采用整体和细胞实验，研究中缝核NG2细胞与5-HT递质和受体及受体后信号的功能关系，NG2细胞在睡眠觉醒神经网络调节中的作用，观察酸枣仁汤对NG2细胞功能变化的干预作用，为新型失眠药物研发提供重要科学依据。

失眠症是一种影响健康最常见的疾病，其研究及治疗对人们的健康至关重要。5-HT是调节睡眠觉醒重要的神经递质。NG2细胞是一种新型的胶质细胞，与神经元形成突触并对脑内环境变化作出快速反应。前期的研究结果表明5-HT含量下降能激活大鼠中缝核星形胶质细胞和小胶质细胞，改变皮质ATP含量和蛋白激酶活性，脑内5-HT与NG2细胞相互作用与相关睡眠调节物质的关系如何？NG2细胞在睡眠觉醒周期中的作用是什么值得进一步研究。本项目采用组织化学法研究了皮层NG2细胞与5-HT、GABA及Glu能神经元的共存关系，采用NG2抗体，研究中缝核NG2细胞在睡眠和觉醒中的作用及与Glu及GABA神经递质含量变化的关系；体外培养NG2细胞，采用相应的激动剂和拮抗剂，研究NG2细胞与5-HT系统的功能关系及受体后信号通路cAMP/Epac /Rap1在其中的作用；同时研究了酸枣仁汤对NG2细胞的作用机制。.研究发现：1. NG2与5-HT、Glu、GABA能神经元在大脑皮层存在不同程度共定位，NG2与5-HT及Glu的共定位情况优于NG2与GABA的共定位；NG2细胞与GABA能神经元的共定位优于NG2与GFAP的共定位。2 .中缝核NG2细胞能调节GABA和Glu的含量。3. NG2细胞能调节大鼠睡眠和觉醒时间，影响EEG波形特征。4. 5-HT含量变化能影响NG2细胞分泌IL-1β、TNF-α、BDNF，NG2细胞上可表达5-HT1A、5-HT2A、5-HT3、5-HT5A、5-HT6和5-HT7受体，5-HT可作用于NG2细胞上5-HT1A、5-HT2A、5-HT3、5-HT6受体影响NG2细胞分泌功能；受体后信号通路PLC和cAMP/Epca/Rap1参与了NG2细胞分泌IL-1β、TNF-α、BDNF。5. 脑内5-HT含量下降可激活皮质和脑干NG2细胞，NG2、GFAP的蛋白表达量明显增加，同时VGluT2蛋白表达升高，TPH、Gephyrin、GABA蛋白表达下降，酸枣仁汤对上述变化具有干预作用。酸枣仁汤含药血清可通过cAMP/Epca/Rap1信号通路调节NG2细胞分泌IL-1β、TNF-α、BDNF。.本研究探讨了NG2细胞与5-HT系统相互作用和机制，酸枣仁汤对PCPA致失眠大鼠NG2细胞活动变化的影响和NG2细胞释放睡眠调节相关活性物质的影响，为新型失眠药研发提供了实验依据。