新化合物YZG-331镇静催眠作用机制的研究

YZG-331 is an analog of N6-(4-hydroxybenzyl) adenine riboside，which was originally isolated from Gastrodia elata Blume. Our laboratory has previously demonstrated that it can produce strong sedative and hypnotic effects. But its mechanism remains to be determined. The use and structure of YZG-331 has been authorized by the national invention patent. Sleep structure analyses were performed by EEG studies in mice. YZG-331 significantly shortened the latency to NREM sleep, and increased the amount of NREM sleep without changing REM sleep. YZG-331 has week affinity with both adenosine A1 and A2A receptors; also it can increase GABA level in mouse brain. In this program, we intends to further explore the sedative and hypnotic mechanisms of compound YZG-331 through neurotransmitters, CaMKII signaling pathway and protein Homer1a. First ,measure GABA and Glu contents in specific mouse brain regions by HPLC-EC after pre-column derivatization with opthaldialdehyde. Cerebral microdialysis is utilized to determine extracellular GABA and Glu levels. The GABA level is regulated by two enzymes, glutamic acid decarboxylase (GAD) and GABA transaminase (GABA-T). Their activities will be detected if necessary. Calcium/calmodulin-dependent protein kinase II (CaMKII) is an important member of calcium/calmodulin-activated protein kinase family, functioning in sleep-wake regulation. The levels of p-CaMKII, PKA and Synapsin I phosphorylation are quantified via Western blot to explore if CaMKII signal pathway involved in the sedative and hypnotic mechanisms of YZG-331. Homer1a plays an important role of the circle of sleep and wake. The levels of Homer1a are tested by Western blot.And the animals with some genes knocked out are used to determin the receptor of YZG-331.In summary, the aim of this program is to find out the targets and understand the neurochemicahl mechanisms of YZG-331 on sedative and hypnotic effects.

以传统安神中药天麻中微量强镇静催眠有效成分为先导物，经结构优化合成获得新结构化合物YZG-331，有较强镇静催眠活性，已获国家发明专利授权。灌胃给YZG-331有明显的中枢镇静催眠作用，能显著增加非快动眼睡眠但不影响快动眼睡眠、药效持续时间适度、肌松作用轻、降压幅度小，毒性小。YZG-331对腺苷A1和A2A受体亲和力较弱，能明显增加小鼠脑内GABA含量，其镇静催眠作用不能被GABAa受体拮抗剂阻断。拟采用微透析-HPLC-ECD检测YZG-331对动物脑内多种神经递质的影响，阐明其镇静催眠作用的神经化学机制，探究YZG-331引起的单胺类神经递质和腺苷变化与Homer1a蛋白的关系， 以及钙调蛋白激酶通路在其中的作用，并用基因条件性敲除小鼠明确YZG-331镇静催眠作用的靶点。研究结果将为开发新型镇静催眠药提供重要依据。

以传统安神中药天麻中微量强镇静催眠有效成分为先导物，经结构优化合成获得新结构化合物YZG-331，前期研究结果显示YZG-331具有明显的镇静催眠活性，但其作用机制尚不清楚。本项目研究了YZG-331发挥镇静催眠作用的神经化学机制、作用靶点及其对脑内睡眠相关因子的调控。YZG-331的镇静催眠作用可以被腺苷受体拮抗剂阻遏，包括非选择性腺苷受体拮抗剂氨茶碱、腺苷A1R拮抗剂DPCPX和A2aR拮抗剂SCH58261。敲降小鼠脑内腺苷A1R或A2aR后YZG-331对小鼠自主活动的抑制作用消失。YZG-331可以降低小鼠下丘脑胞内钙离子水平及其下游因子CaMKII的磷酸化水平，YZG-331对p-CaMKII的调控可被DPCPX和SCH58261阻断，提示腺苷A1、A2a受体介导了YZG-331的镇静催眠作用，腺苷受体后Ca2+-CaMKII通路是YZG-331发挥镇静催眠作用的机制之一。神经化学机制研究结果显示YZG-331可以显著升高小鼠多个脑区突触间隙中GABA和Ala水平，降低单胺类神经递质DA和5-HT水平，这些变化能够导致大脑皮层兴奋性的下降。此外，YZG-331可使NMDAR的NR2B亚基磷酸化增加，导致其与细胞膜内PSD95的相互作用减弱，从而促进NMDAR的胞吞作用，使NMDAR内化增多，膜表面数量减少，NMDAR介导的皮层神经元兴奋性明显下降，这可能是YZG-331镇静催眠作用的另一机制。另外，我们发现YZG-331可以有效延长硝酸士的宁引起的小鼠惊厥潜伏期，同时降低死亡率。综上研究结果，YZG-331镇静催眠作用的产生是多条通路共同作用的结果。YZG-331镇静催眠机制的阐明以及抗惊厥作用的发现为将YZG-331开发成新型镇静催眠药提供了重要依据。

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