化合物B2镇静催眠作用机制的研究

发布时间：2018-07-14 14:40

【摘要】：以传统安神中药天麻中微量强镇静催眠有效成分N6-对羟苄基腺苷为先导化合物,经150余个化合物的合成优化获得新结构化合物B2,对小鼠有很强的中枢镇静催眠作用。本研究拟进一步从GABA能神经系统、单胺能神经系统和钙调蛋白激酶通路三个方面探究B2的镇静催眠机制。 GABA是脑内主要的抑制性氨基酸类神经递质,Glu是脑内重要的兴奋性神经递质,均与睡眠-觉醒关系密切。因此,我们首先运用OPA柱前衍生化HPLC-EC法测定了B2对小鼠不同脑区GABA和Glu含量的影响。结果表明,B2(5mg/kg,i.p.)可使小鼠下丘脑中GABA含量显著升高39%,大脑皮层中GABA含量显著升高32%：同时使下丘脑中Glu含量显著降低22%,大脑皮层中Glu含量显著降低21%。下丘脑腹外侧视前区(ventrolateral preoptic area, VLPO)是重要的促睡眠中枢,通过投射到促觉醒中枢结节乳头体核(tuberomammillary nucleus, TMN)的神经末梢释放GABA而诱导睡眠。因此,我们采用微透析法进一步对给予B2后小鼠TMN区细胞外液GABA的水平进行了测定。结果表明,小鼠腹腔注射B2(5mg/kg)10mmin后引起TMN区细胞外液GABA水平升高至给药前的1.7倍。小鼠脑内GABA含量主要受GABA合成酶GAD和GABA水解酶GABA-T的调控。检测结果表明,B2(5mg/kg,i.p.)可使小鼠下丘脑及大脑皮层的GAD酶活性分别升高43%和31%,但对GABA-T酶的活性无显著影响。小鼠腹腔注射GAD酶抑制剂盐酸氨基脲(semicarbazlde hydrochloride, SCZ)(100mg/kg, i.p.)对戊巴比妥钠(40mg/kg,i.p.)诱导的小鼠睡眠潜伏期和睡眠时间无明显影响,但能明显抑制B2(1和5mg/kg,i.p.)对小鼠睡眠的延长作用。通过对EEG信号分析表明,化合物B2(5mg/kg, i.p.)可显著缩短小鼠睡眠潜伏期,该作用不能被GAD酶抑制剂SCZ所拮抗。SCZ(100mg/kg, i.p.)本身对小鼠睡眠结构无显著影响,但它可以显著抑制B2(5mg/kg,i.p.)的促睡眠作用。提示B2可激活GAD酶,进而增加脑内GABA的含量。 单胺类神经递质NE、DA和5-HT等均参与睡眠-觉醒的调节。我们采用HPLC-EC法检测了B2对小鼠不同脑区组织中NE、DA和5-HT等单胺类神经递质水平的影响。结果表明,与对照组相比,B2(5mg/kg,i.p.)使小鼠下丘脑中NE含量显著降低19%；使纹状体中DA含量显著降低33%；使下丘脑中5-HT水平显著降低46%。小鼠脑内DA及5-HT的代谢主要由MAO调控。结果表明,与对照组相比,B2(5mg/kg, i.p.)组小鼠下丘脑及纹状体中MAO活性有升高的趋势。提示B2可能不仅仅是通过调控MAO的活性而影响小鼠脑内单胺类神经递质的水平,也可能通过其他途径发挥作用。 钙离子/调素依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinase Ⅱ, CaMKⅡ是钙离子/调素依赖的蛋白激酶家族成员,在睡眠-觉醒调节中具有重要作用。我们运用Western blot法对p-CaMKⅡ的表达水平进行了测定。结果表明,给予B2(5mg/kg, i.p.)15min后可引起小鼠下丘脑p-CaMKⅡ水平显著降低49%。鉴于侧脑室注射CaMKⅡ磷酸化抑制剂KN93也可降低小鼠下丘脑区p-CaMKⅡ表达水平并显著抑制小鼠的自主活动,我们推测B2抑制小鼠下丘脑区CaMKⅡ磷酸化可能是其镇静催眠的作用机制之一。为了初步探究CaMKⅡ信号通路介导B2镇静催眠作用的机制,我们分别对其上下游的相关蛋白PKA及Synapsin Ⅰ的磷酸化水平进行了测定。结果表明,B2(5mg/kg, i.p.)对p-PKA的表达无显著影响,但可显著降低小鼠下丘脑p-Synapsin Ⅰ的表达水平,提示B2可能通过抑制CaMKⅡ磷酸化并进一步抑制Synapsin Ⅰ磷酸化而发挥镇静催眠作用。 综上所述,新型镇静催眠化合物B2的镇静催眠机制可能为：一、激活GAD酶,使GABA的合成增加,进而通过受体后效应发挥镇静催眠作用；二、降低相关脑区单胺类神经递质的水平,进而发挥镇静催眠作用；三、抑制CaMKⅡ磷酸化,进而抑制Synapsin Ⅰ磷酸化,减少兴奋性神经递质的释放而发挥镇静催眠作用。
[Abstract]:In this study , the sedative hypnotic mechanism of B2 was studied in three aspects : GABA - ergic nervous system , single - amine - energy nervous system and calcium - regulated protein kinase pathway .

The effect of B2 ( 5 mg / kg , i . p . ) on the level of GABA and Glu in the hypothalamus and cerebral cortex of mice was significantly reduced . The results showed that B2 ( 5 mg / kg , i . p . ) could significantly decrease the level of GABA and Glu in the hypothalamus and cerebral cortex of mice .

The effects of B2 ( 5 mg / kg , i.p . ) on the levels of NE , DA and 5 - HT in different brain regions of mice were detected by HPLC - EC . The results showed that the content of NE , DA and 5 - HT in the hypothalamus of mice was significantly decreased by 19 % compared with the control group .
the content of DA in corpus striatum was significantly reduced by 33 % ;
The level of 5 - HT in hypothalamus was significantly decreased by 46 % . The metabolism of DA and 5 - HT in brain of mice was mainly regulated by MAO . The results showed that the activity of MAO in the hypothalamus and striatum of mice in B2 ( 5 mg / kg , i . p . ) group was higher than that of control group .

In order to investigate the mechanism of CaMK鈪，

本文编号：2121971