Sedative And Hypnotic Mechanisms Of Compound B2

N6-(3-methoxyl-hydroxybenzyl) adenine riboside (B2) is one of the150analogs of N6-(4-hydroxybenzyl) adenine riboside which was an active compound originally isolated from Gastrodia elata Blume. Previous study demonstrated that B2can produce strong sedative and hypnotic effects but the mechanisms remain to be clarified. This study intends to further explore the sedative and hypnotic mechanisms of compound B2from three aspects, including the effect of B2on the GABAergic system, the effect of B2on monoamine neurotransmitters and the effect of B2on the CaMKII signaling pathway.GABA is the primary inhibitory neurotransmitter in the CNS, whereas Glu is the most abundant excitatory neurotransmitter. They play an important role in sleep-wake regulation. We first measured GABA and Glu contents in different mouse brain regions by HPLC-EC after pre-column derivatization with opthaldialdehyde. The results showed that B2significantly increased the GABA level by39%in mouse hypothalamus and by32%in mouse cortex, and decreased Glu content by22%in mouse hypothalamus and by21%in mouse cortex. The ventrolateral preoptic nucleus (VLPO) is a group of neurons in the hypothalamus 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 result showed that the extracellular GABA level in mouse TMN was significantly increased after10min administration of B2(5mg/kg, i.p.), and the GABA level was1.7fold to the baseline value. The GABA level is regulated by two enzymes, glutamic acid decarboxylase (GAD) and GABA transaminase (GABA-T). The results showed that B2(5mg/kg, i.p.) significantly increased the GAD activity in mouse hypothalamus. However, it had no influence on the GABA-T activity. B2(5mg/kg, i.p.) significantly prolonged the duration of loss of righting reflex induced by pentobarbital sodium (40mg/kg, i.p.). The GAD inhibitor SCZ (100mg/kg, i.p.) had no influence on sleep latency and sleep duration in pentobarbital sodium treated mice when it was used alone, respectively. However, SCZ could block the potentiating effect of B2on pentobarbital sodium-induced sleep in mice. Sleep structure analyses were performed by EEG studies in mice. The result confirmed B2significantly shortened the latency to NREM sleep, and increased the amount of NREM sleep; GAD enzyme inhibitor SCZ blocked the sleep potentiating effect of B2. Based on these findings, it suggests that activation of GAD contributed to the increase of GABA content in mouse hypothalamus.Monoamine neurotransmitters are closely associated with the sleep-wake regulation. We detected the contents of NE, DA and5-HT by HPLC-EC. The results showed that B2(5mg/kg, i.p.) could significantly reduce the hypothalamus NE content by19%and reduce DA content in the striatum by33%.5-HT content in hypothalamus was significantly decreased by46%. The metabolism of DA and5-HT are primarily regulated by monoamine oxidase (MAO) in mouse brain, so we further determined the effect of B2on the MAO activity. The effect of compound B2on MAO activities in mouse hypothalamus, striatum and cortex were detected by colorimetric method. There was an increasing trend in MAO activities in mouse hypothalamus and striatum after administration of B2(5mg/kg, i.p.). These results suggest that the decrease of monoamine contents in different mouse brain regions induced by B2might be not only mediated by regulating the MAO acticity, but also mediated by other ways.Calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) is an important member of calcium/calmodulin-activated protein kinase family, functioning in the sleep-wake regulation. We quantified the level of p-CaMKⅡ expression by Western blot. The result showed that B2significantly decreased the level of p-CaMKⅡ expression in mouse hypothalamus15min after the administration. Given that KN93, an inhibitor of CaMKⅡ phosphorylation, could also decrease the level of p-CaMKⅡ expression in mouse hypothalamus and inhibit the locomotor activity of mice. These findings indicate B2may be inducing sedative and hypnotic effects by down regulating the p-CaMKⅡ expression in mouse hypothalamus. In order to explore the sedative and hypnotic mechanisms of B2mediated by CaMKII signal pathway, the PKA and Synapsin Ⅰ phosphorylation expression levels were determined. The results showed that B2(5mg/kg, i.p.) had no significant effect on the p-PKA expression, but can significantly reduce the p-Synapsin I expression level in mouse hypothalamus, suggesting that B2may exert sedative and hypnotic effects by suppressing p-Synapsin I expression via inhibiting CaMKII phosphorylation.In summary, the sedative and hypnotic mechanisms of compound B2might attribute to:First, B2activates GAD enzyme which leads to GABA increase and then exerts sedative and hypnotic effects through relevant receptor. Second, B2reduces the monoamine neurotransmitter contents which promote arousal in mouse brain, and then induces sedative and hypnotic effects. Third, B2inhibits phosphorylation of CaMKII, then inhibits the phosphorylation of Synapsin I, which may reduce excitatory neurotransmitter release and then exert sedative and hypnotic effects.