Hypnotic Effects And Mechanisms Of Novel Modifiers Of N~6-(4-hydroxybenzyl) Adenosine Riboside:YZG-331

Professor SHI Jiangong in the department of Phytochemistry isolated the new compound NHBA.We got a pair of enantiomer YZG-330/YZG331 via modifying this leading structure.The purpose of this study was to investigate the sedative and hypnotic effects of YZG-331 and its mechanism.In the open field experiment,YZG-331 inhibited the locomotor activity in a dose-dependent manner.Administrated orally at the dose of 10 and 50 mg/kg,YZG-331 decreased the total locomotor activity by 81.0%(P<0.001)and 89.2%(P<0.001)respectively compared with vehicle.The hypnotic effect was evaluated by the latency and duration of the loss of righting reflex(LORR)in the threshold dosage of sodium pentobarbital treated mice.YZG-331(5,10,20 mg/kg i.g.)increased the sleeping time by 35.3%(P<0.01),71.2%(P<0.001),101.7%(P<0.001),respectively.YZG-331(1,5 and 25 mg/kg,i.g.)increased the sleep rate induced by subthreshold dosage of sodium pentobarbital(25 mg·kg-1,i.p.)in mice by 0%,75%(P<0.01)and 100%(P<0.01).GABAA receptor is directly related to sleep,including five binding sites.Flumazenil is the antagonist of BZ site,and the bicuculline is the antagonist of GABA site.However,neither Flumazenil nor the bicuculline can antagonize the sedative effect of YZG-331.Both the sites cannot combine with YZG-331.GABA is the major inhibitory neurotransmitter in the brain,and Glu is an important excitatory neurotransmitter.Glu is a precursor of the GABA,GABA can be generated under the catalysis of GAD.It is important to the sleep and wake regulation.We first measured the contents of GABA and Glu in cortex and hypothalammus by HPLC-ECD after pre-column derivatization with OPA.The results showed that YZG-331 significantly increased the GABA level by 10.3%in mouse hypothalamus and by 11.6 in mouse cortex.The ventrolateral preoptic nucleus(VLPO)is a primary area to regulate the sleep.The neurons in the VLPO that can release the inhibitory neurotransmitter GABA into its projection area tuberomammillary nucleus(TMN)to promote sleep.We measured extracellular GABA level in mouse TMN by cerebral microdialysis.The level of GABA was significantly increased after administration of YZG-331(40 mg·kg-1,i.g.).After 20 min administration of YZG-331,the extracellular GABA level in mouse TMN was 2.3 fold to the basal value.The GABA level is regulated by GAD and GABA-T.GABA can be generated under the catalysis of GAD.GABA-T catalyzes GABA hydrolysis.We measured the activity of the GAD and GABA-T.The resluts showed that YZG-331(40 mg·kg-1,i.g.)significantly increased the GAD activity of cerebral cortex by 106.8%(P<0.05).The GAD activity of the hypothalamus was increased by 58.6%in the YZG-331 group.However,it had no influence on the GABA-T activity.SCZ(100 mg·kg-1,i.g.),the GAD inhibitor,had no influence in pentobarbital sodium treated mice when it was used alone,respectively.However,SCZ could decrease the sleeping time of YZG-331 on penbarbital sodium-induced sleep in mice.SCZ didn’t influnce the architecture of sleep significantly.EEG results showed that YZG-331 significantly increased the amount of NREM sleep.The amount of REM sleep and wake are decreased.Monoamine neurotransmitters can regulate the cycle of sleep and wake.The contents of DA and 5-HT were detected by HPLC-ECD.The results showed that YZG-331(40 mg·kg-1,i.g.)could significantly increase the hypothalamus DA content to 40.4%(P<0.01),DOPAC content in hypothalamus was increased to 103%(P<0.01).The metabolism of monoamine nertotransmitters DA and 5-HT are primarily regulated by MAO,so we detected the activity of MAO after the adminstration of YZG-331.The effect of YZG-331 on MAO activities in mouse hupothalamus and cortex were detected by colorimetric method.The activity of MAO in the hypothalamus increased significantly after 30 min adminstion of YZG-331.However,YZG-331 had no influence on the MAO activity in cortex.These results suggest that the change of monoamine contents in different mouse brain regions might be not only mediated by regulating the MAO activity,but also mediated by other ways.In summary,the sedative and hypnotic mechanism of compound YZG-331 might attribute to:YZG-331 activates GAD enzyme which leads to GABA increase.More GABA combine with the GABAA receptor.The early stage of the laboratory experiments show that YZG-331 can increase the concentration of chloride ion in cells.The nerve cells can be inhibited.So the YZG-331 can promote sleep.