發(fā)布時(shí)間：2018-07-14 14:40

【摘要】：以傳統(tǒng)安神中藥天麻中微量強(qiáng)鎮(zhèn)靜催眠有效成分N6-對(duì)羥芐基腺苷為先導(dǎo)化合物,經(jīng)150余個(gè)化合物的合成優(yōu)化獲得新結(jié)構(gòu)化合物B2,對(duì)小鼠有很強(qiáng)的中樞鎮(zhèn)靜催眠作用。本研究擬進(jìn)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(gè)方面探究B2的鎮(zhèn)靜催眠機(jī)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(yùn)用OPA柱前衍生化HPLC-EC法測(cè)定了B2對(duì)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(shí)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(dǎo)睡眠。因此,我們采用微透析法進(jìn)一步對(duì)給予B2后小鼠TMN區(qū)細(xì)胞外液GABA的水平進(jìn)行了測(cè)定。結(jié)果表明,小鼠腹腔注射B2(5mg/kg)10mmin后引起TMN區(qū)細(xì)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(cè)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(duì)GABA-T酶的活性無(wú)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(duì)戊巴比妥鈉(40mg/kg,i.p.)誘導(dǎo)的小鼠睡眠潛伏期和睡眠時(shí)間無(wú)明顯影響,但能明顯抑制B2(1和5mg/kg,i.p.)對(duì)小鼠睡眠的延長(zhǎng)作用。通過對(duì)EEG信號(hào)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(duì)小鼠睡眠結(jié)構(gòu)無(wú)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(jìn)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(cè)了B2對(duì)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(duì)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(duì)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(shì)。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(yùn)用Western blot法對(duì)p-CaMKⅡ的表達(dá)水平進(jìn)行了測(cè)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(dá)水平并顯著抑制小鼠的自主活動(dòng),我們推測(cè)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(jī)制之一。為了初步探究CaMKⅡ信號(hào)通路介導(dǎo)B2鎮(zhèn)靜催眠作用的機(jī)制,我們分別對(duì)其上下游的相關(guān)蛋白PKA及Synapsin Ⅰ的磷酸化水平進(jìn)行了測(cè)定。結(jié)果表明,B2(5mg/kg, i.p.)對(duì)p-PKA的表達(dá)無(wú)顯著影響,但可顯著降低小鼠下丘腦p-Synapsin Ⅰ的表達(dá)水平,提示B2可能通過抑制CaMKⅡ磷酸化并進(jìn)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(jī)制可能為：一、激活GAD酶,使GABA的合成增加,進(jìn)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(jìn)而發(fā)揮鎮(zhèn)靜催眠作用；三、抑制CaMKⅡ磷酸化,進(jìn)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(hào)：2121971