从大脑皮质ATP/P2X受体途径探讨PCPA所致失眠的分子机制及酸枣仁汤的干预研究

Insomnia is one of the most common diseases affecting health. Glial cells play an important role in the processes of sleep regulation; current research shows that ATP is a major astrocytic gliotransmitter in vivo; it is a significant source of extracellular adenosine in the brain; and it can act on purinergic receptors by activating different intracellular signal transduction pathways, thereby regulating sleep activity. The PCPA insomnia animal model is commonly used in insomnia drug research; preliminary studies find that PCPA insomnia and glial cell activation are closely related, but whether PCPA insomnia causes changes in the cerebral cortex ATP level, and the concomitant effects of such changes are unclear. This study uses the established PCPA insomnia model, high performance liquid chromatography and double immunofluorescence staining, confocal laser scanning microscopy, and Western blotting and RT-PCR methods to observe ATP concentration in the cerebral cortex, and the effects of PCPA and Suanzaoren decoction on purine receptors and post-receptor signaling pathways. This study will clarify the signal transduction mechanisms in PCPA insomnia, revealing potential targets of Suanzaoren decoction in the treatment of insomnia. It seeks to further clarify the link targets of sleep drug action, provided 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。ATP及嘌呤P2受体在睡眠调控过程中起到了重要作用。目前认为，神经胶质细胞可释放ATP等递质介导神经系统的信息传递，ATP可作用于嘌呤P2受体，激活不同的细胞内信号转导通路，进而调节睡眠活动。PCPA失眠动物模型是常用的睡眠药物研究的载体，前期研究发现PCPA失眠与神经胶质细胞激活有密切关系，但PCPA失眠是否导致大脑皮质ATP含量变化并将该作用引向何方尚不清楚。本研究拟通过建立PCPA失眠模型，运用高效液相色谱法、免疫荧光双染法、激光共聚焦、Western blotting和RT-PCR等方法，观察PCPA失眠模型中ATP、嘌呤P2X受体及受体后信号活动变化等的影响。本研究将阐明PCPA所致失眠的信号传导机制，揭示酸枣仁汤治疗失眠症的潜在靶点，有利于进一步明确促眠药物作用的环节、作用的靶点，为促眠药物的研发提供分子生物学证据，为失眠症的防治提供新的思路。

失眠症是一种影响健康最常见的疾病。脑内5-HT失调是形成失眠的重要原因之一，但脑内5-HT改变后通过何种途径引起大脑皮质兴奋性变化目前尚未明确。PCPA选择性抑制色氨酸羟化酶活性而抑制5-HT合成，造成动物失眠，是常用的失眠模型。前期研究发现PCPA失眠与神经胶质细胞激活有密切关系，但PCPA失眠是否导致大脑皮质ATP含量变化并将该作用引向何方尚不清楚。本研究通过建立PCPA失眠模型，运用高效液相色谱法、免疫荧光双染法、分子生物学等方法，研究了PCPA失眠模型中ATP含量变化 、嘌呤P2X受体及受体后信号通路蛋白和基因表达的变化和酸枣仁汤的干预作用。研究结果发现① PCPA失眠后第4、5、6、8、10天大鼠皮质组织ATP含量下降、Na+/K+-ATPase和Ca2+/Mg2+-ATPase的活性明显下降，SZRD能调节PCPA失眠大鼠皮质ATP酶的活性，升高ATP水平。② PCPA失眠大鼠皮质GFAP和P2X7R蛋白表达增强，P2X7R基因mRNA表达上调，该效应持续到10天仍保持高水平，SZRD能明显下调GFAP及P2X7R基因mRNA和蛋白的表达。③ PCPA能导致大鼠皮质P38MAPK蛋白和基因mRNA表达增加，而PKC和CAMKⅡ的蛋白和基因mRNA表达水平下降，SZRD能对这些变化产生拮抗作用；④ P2X受体非特异性拮抗剂（OxATP）及P2X7受体高选择性拮抗剂（A438079）能使P38MAPK蛋白和基因mRNA表达下降，PKC和CAMKⅡ的蛋白及基因mRNA表达水平上升；而P2X7受体选择性激动剂（BzATP）引起P38MAPK蛋白和基因mRNA表达水平升高，PKC和CAMK Ⅱ的蛋白表达和基因mRNA表达水平下降。结果表明P2X7 受体介导了脑内5-HT下降所致的前额皮质受体后信号P38MAPK、PKC和CAMKⅡ蛋白和基因表达的变化。使用酸枣仁汤干预后，OxATP及A438079所致的PKC、CaMKⅡ、P38MAPK蛋白及基因mRNA表达变化不再出现，但BzATP所致的P38MAPK蛋白和基因mRNA表达水平升高、PKC和CAMK Ⅱ的蛋白表达和基因mRNA表达水平下降的效应依然存在，该结果提示SZRD可能激活P2X受体。.本研究为新型促眠药物的研发提供了实验依据，为失眠症的防治提供了新的思路。