Study On Sedative And Hypnotic Mechanisms Of The Novel Compound YZG-331

The compound YZG-33 1 is from Prof.Shi Jiangong’s group of Institute of Materia Medica,Chinese Academy of Medical Sciences.Through the close cooperation of medicinal chemistry and pharmacology,researchers discovered the trace active ingredient in Chinese traditional medicine Gastrodia elata,and after further structural modification,a novel compound YZG-331 is obtained.Its structure is shown in Figure 1.Earlier studies in this laboratory found that intragastric administration of YZG-33 1(1.25-40 mg·kg-1)can dose-dependently inhibited the autonomous activity of mouse and it can significantly prolonged the sleep time and shortened the sleep latency induced by suprathreshold sodium pentobarbital,and increased the subthreshold dose of sodium pentobarbital-induced sleep onset in mice.So it is of great significance to illustrate its mechanism for the development of new sedative and hypnotic drugs.The animal experiments in this study were all approved by the Animal Ethics Committee of the Institute of Materia Medica,Chinese Academy of Medical Sciences.In this experiment,Western blot technology was used to detect the expression of NMDAR on the cell membrane and endosome in the frontal cortex ofmice,and protein immunoprecipitation technology was used to explore the interactions of NMDAR endocytosis-related proteins.High performance liquid chromatography(HPLC)detection technology was used to detect the content of y-aminobutyric acid(GABA),glutamate and GABA synthetase—glutamate decarboxylase activity in tissue homogenate of sleep-related brain regions in mice.At the same time,the acetylcholine detection kit and microplate reader method were used to detect the content of acetylcholine in the homogenate of these brain regions.In addition,this study evaluated the sedative and hypnotic effects of 22 metabolites of compound YZG-331 using the sleep time-prolonging test with threshold dosage pentobarbital sodium.The results showed that the expression levels of NMDAR subunits NR1 and NR2B on the cell membrane of the frontal cortex in mice were down-regulated after administration of compound YZG-331(40 mg·kg-1),and compared with control group,the content change of NR2B had statistical difference(p<0.05).At the same time,the expression levels of NR1 and NR2B in frontal cortex cell endosome increased significantly(p<0.05,p<0.01).Western blot results showed that the postsynaptic density-95(PSD95)expression significantly reduced in co-immunoprecipitation complex precipitated with anti-NR2B as bait protein in mouse prefrontal cortex(p<0.01).After intragastric administration of compound YZG-331(40 mg·kg-1)for 15 min,there was no significant changes in the contents of GABA and glutamate in the mouse cortex,hypothalamus,thalamus,and brainstem tissue homogenates.After intragastric administration of compound YZG-331(10,40 mg·kg-1)for 15 min,the results showed that it had no significant effect on GABA synthase—glutamate decarboxylase activity in the mouse cortex and hypothalamus.The detection results of acetylcholine content showed that after intragastric administration of compound YZG-331(40 mg·kg-1)for 15 min,the content of acetylcholine in mouse hypothalamus increased by 20.2%(p<0.05),but there was no significant changes in cortex,striatum,hippocampus and basal forebrain.The results of the sleep time-prolonging test with threshold dosage pentobarbital sodium in mice showed that YZG-331’ metabolites Xcb-3004,Xcb-3014,Xcb-3020,Xcb-3 024 and Xcb-3025 shortened the sleep latency and increased the sleep time of mice.In summary,the compound YZG-331 reduces the expressions of NR1 and NR2B on the cell membrane and increases their contents in the endosome by disrupting the binding ability of the NMDAR subunit NR2B and PSD95 in the post-synaptic compact.The membrane expressions of NMDA receptors are reduced,which weakens the signal conduction of excitatory neurotransmitter Glu,which may be one of the mechanisms for its sedative and hypnotic effects.YZG-331 has no significant effects on the contents of GABA and glutamate,so does glutamate decarboxylase activity in mouse brain homogenates,indicating that the sedative and hypnotic effects of YZG-331 may not be caused by the anabolism of the neurotransmitter GABA.In addition,the effect of YZG-33 1 on the content of acetylcholine in various brain regions of mouse may be related to its effect on learning and memory in mice.