發(fā)布時間：2018-07-14 14:40

【摘要】：以傳統(tǒng)安神中藥天麻中微量強鎮(zhèn)靜催眠有效成分N6-對羥芐基腺苷為先導化合物,經(jīng)150余個化合物的合成優(yōu)化獲得新結(jié)構(gòu)化合物B2,對小鼠有很強的中樞鎮(zhèn)靜催眠作用。本研究擬進一步從GABA能神經(jīng)系統(tǒng)、單胺能神經(jīng)系統(tǒng)和鈣調(diào)蛋白激酶通路三個方面探究B2的鎮(zhèn)靜催眠機制。 GABA是腦內(nèi)主要的抑制性氨基酸類神經(jīng)遞質(zhì),Glu是腦內(nèi)重要的興奮性神經(jīng)遞質(zhì),均與睡眠-覺醒關(guān)系密切。因此,我們首先運用OPA柱前衍生化HPLC-EC法測定了B2對小鼠不同腦區(qū)GABA和Glu含量的影響。結(jié)果表明,B2(5mg/kg,i.p.)可使小鼠下丘腦中GABA含量顯著升高39%,大腦皮層中GABA含量顯著升高32%：同時使下丘腦中Glu含量顯著降低22%,大腦皮層中Glu含量顯著降低21%。下丘腦腹外側(cè)視前區(qū)(ventrolateral preoptic area, VLPO)是重要的促睡眠中樞,通過投射到促覺醒中樞結(jié)節(jié)乳頭體核(tuberomammillary nucleus, TMN)的神經(jīng)末梢釋放GABA而誘導睡眠。因此,我們采用微透析法進一步對給予B2后小鼠TMN區(qū)細胞外液GABA的水平進行了測定。結(jié)果表明,小鼠腹腔注射B2(5mg/kg)10mmin后引起TMN區(qū)細胞外液GABA水平升高至給藥前的1.7倍。小鼠腦內(nèi)GABA含量主要受GABA合成酶GAD和GABA水解酶GABA-T的調(diào)控。檢測結(jié)果表明,B2(5mg/kg,i.p.)可使小鼠下丘腦及大腦皮層的GAD酶活性分別升高43%和31%,但對GABA-T酶的活性無顯著影響。小鼠腹腔注射GAD酶抑制劑鹽酸氨基脲(semicarbazlde hydrochloride, SCZ)(100mg/kg, i.p.)對戊巴比妥鈉(40mg/kg,i.p.)誘導的小鼠睡眠潛伏期和睡眠時間無明顯影響,但能明顯抑制B2(1和5mg/kg,i.p.)對小鼠睡眠的延長作用。通過對EEG信號分析表明,化合物B2(5mg/kg, i.p.)可顯著縮短小鼠睡眠潛伏期,該作用不能被GAD酶抑制劑SCZ所拮抗。SCZ(100mg/kg, i.p.)本身對小鼠睡眠結(jié)構(gòu)無顯著影響,但它可以顯著抑制B2(5mg/kg,i.p.)的促睡眠作用。提示B2可激活GAD酶,進而增加腦內(nèi)GABA的含量。 單胺類神經(jīng)遞質(zhì)NE、DA和5-HT等均參與睡眠-覺醒的調(diào)節(jié)。我們采用HPLC-EC法檢測了B2對小鼠不同腦區(qū)組織中NE、DA和5-HT等單胺類神經(jīng)遞質(zhì)水平的影響。結(jié)果表明,與對照組相比,B2(5mg/kg,i.p.)使小鼠下丘腦中NE含量顯著降低19%；使紋狀體中DA含量顯著降低33%；使下丘腦中5-HT水平顯著降低46%。小鼠腦內(nèi)DA及5-HT的代謝主要由MAO調(diào)控。結(jié)果表明,與對照組相比,B2(5mg/kg, i.p.)組小鼠下丘腦及紋狀體中MAO活性有升高的趨勢。提示B2可能不僅僅是通過調(diào)控MAO的活性而影響小鼠腦內(nèi)單胺類神經(jīng)遞質(zhì)的水平,也可能通過其他途徑發(fā)揮作用。 鈣離子/調(diào)素依賴性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinase Ⅱ, CaMKⅡ是鈣離子/調(diào)素依賴的蛋白激酶家族成員,在睡眠-覺醒調(diào)節(jié)中具有重要作用。我們運用Western blot法對p-CaMKⅡ的表達水平進行了測定。結(jié)果表明,給予B2(5mg/kg, i.p.)15min后可引起小鼠下丘腦p-CaMKⅡ水平顯著降低49%。鑒于側(cè)腦室注射CaMKⅡ磷酸化抑制劑KN93也可降低小鼠下丘腦區(qū)p-CaMKⅡ表達水平并顯著抑制小鼠的自主活動,我們推測B2抑制小鼠下丘腦區(qū)CaMKⅡ磷酸化可能是其鎮(zhèn)靜催眠的作用機制之一。為了初步探究CaMKⅡ信號通路介導B2鎮(zhèn)靜催眠作用的機制,我們分別對其上下游的相關(guān)蛋白PKA及Synapsin Ⅰ的磷酸化水平進行了測定。結(jié)果表明,B2(5mg/kg, i.p.)對p-PKA的表達無顯著影響,但可顯著降低小鼠下丘腦p-Synapsin Ⅰ的表達水平,提示B2可能通過抑制CaMKⅡ磷酸化并進一步抑制Synapsin Ⅰ磷酸化而發(fā)揮鎮(zhèn)靜催眠作用。 綜上所述,新型鎮(zhèn)靜催眠化合物B2的鎮(zhèn)靜催眠機制可能為：一、激活GAD酶,使GABA的合成增加,進而通過受體后效應(yīng)發(fā)揮鎮(zhèn)靜催眠作用；二、降低相關(guān)腦區(qū)單胺類神經(jīng)遞質(zhì)的水平,進而發(fā)揮鎮(zhèn)靜催眠作用；三、抑制CaMKⅡ磷酸化,進而抑制Synapsin Ⅰ磷酸化,減少興奮性神經(jīng)遞質(zhì)的釋放而發(fā)揮鎮(zhèn)靜催眠作用。
[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，

本文編號：2121971