羅蕭蕭，趙以林，李世勇，譚蕾，王金韜

(華中科技大學(xué)同濟(jì)醫(yī)學(xué)院第二臨床學(xué)院，武漢 430030)

·藥物研究·

氯胺酮通過(guò)MEF2信號(hào)通路調(diào)節(jié)發(fā)育期神經(jīng)元突觸生長(zhǎng)及突觸重塑*

羅蕭蕭，趙以林，李世勇，譚蕾，王金韜

(華中科技大學(xué)同濟(jì)醫(yī)學(xué)院第二臨床學(xué)院，武漢 430030)

目的 探討氯胺酮不同暴露時(shí)間下對(duì)發(fā)育神經(jīng)元肌細(xì)胞增強(qiáng)因子2(MEF2)信號(hào)通路及突觸生長(zhǎng)相關(guān)蛋白synapsin I表達(dá)影響。方法新生5 d齡SD大鼠,隨機(jī)分為2,4,6和24 h氯胺酮組(T組)和對(duì)照組(C組)。將新生5 d SD大鼠皮下注射氯胺酮(20 mg·kg-1)干預(yù)。對(duì)照組不做任何處理。干預(yù)后在各對(duì)應(yīng)時(shí)間點(diǎn)提取海馬神經(jīng)元總RNA,利用real-time PCR進(jìn)行定量分析MEF2信號(hào)通路(MEF2 mRNA、synGAP I mRNA和Arc mRNA)及突觸形成相關(guān)基因synapsin I mRNA表達(dá)水平。結(jié)果與C組相比,用氯胺酮干預(yù)后時(shí)間依賴(lài)性下調(diào)海馬神經(jīng)元MEF2 mRNA、synGAP I mRNA、Arc mRNA(P＜0.05)。Synapsin I mRNA表達(dá)則時(shí)間依賴(lài)性上調(diào)(P＜0.05)。麻醉后24 h恢復(fù)正常。結(jié)論氯胺酮短暫下調(diào)MEF2信號(hào)通路而上調(diào)synapsin I表達(dá),其機(jī)制可能是Arc表達(dá)上調(diào)增加突觸數(shù)目以致synapsin I表達(dá)上調(diào)。

氯胺酮；海馬；發(fā)育期神經(jīng)元；肌細(xì)胞增強(qiáng)因子2；突觸素

在神經(jīng)元發(fā)育過(guò)程中,軸突的分化生長(zhǎng)以及突觸聯(lián)系形成是神經(jīng)系統(tǒng)功能得以體現(xiàn)的基礎(chǔ)及記憶形成的關(guān)鍵步驟,而突觸素在神經(jīng)元突起的生長(zhǎng)、突觸的形成以及突觸的維持中發(fā)揮重要作用[1-2]。發(fā)育期神經(jīng)元是軸突生長(zhǎng)和突出形成的重要階段,特別容易受到外界因素干擾如氯胺酮等。肌細(xì)胞增強(qiáng)因子2 (myocyte enhancer factor 2,MEF2)作為鈣依賴(lài)性調(diào)節(jié)因子,在神經(jīng)系統(tǒng)突觸發(fā)育和分化中發(fā)揮著重要的作用,特別是下游基因synGAP和Arc在調(diào)控突觸形成過(guò)程中起著重要作用[3-4]。筆者在本研究探討氯胺酮對(duì)發(fā)育期神經(jīng)元突觸素的表達(dá)與MEF2信號(hào)通路的關(guān)系。

1 材料與方法

1.1 試劑與儀器 逆轉(zhuǎn)錄試劑盒(日本Takara公司,批號(hào):D6110A)；Trizol試劑(上海華舜公司,批號(hào): WR202)；實(shí)時(shí)定量PCR試劑盒(日本Takara公司,批號(hào):DRRO81S)；BioPhotometer plus核酸蛋白測(cè)定儀(德國(guó)Ependorf公司)；ABI實(shí)時(shí)熒光定量PCR儀(美國(guó)Life Technologies公司)；氯胺酮注射劑(福建吉田藥業(yè)有限公司,規(guī)格:2 mL∶100 mg,批號(hào):H35010148)。

1.2 動(dòng)物選擇及分組 出生5 d的Sprague-Dawley (SD)大鼠30只,雌雄不拘,體質(zhì)量10～13 g,由華中科技大學(xué)同濟(jì)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供。利用隨機(jī)數(shù)字表將實(shí)驗(yàn)動(dòng)物分為5組(n=6):對(duì)照組(C組)、氯胺酮2 h組、氯胺酮4 h組、氯胺酮6 h組和氯胺酮24 h組,其中氯胺酮各時(shí)間點(diǎn)組統(tǒng)稱(chēng)為T(mén)組。將新生5 d SD大鼠皮下單次注射氯胺酮(20 mg·kg-1)干預(yù)而對(duì)照組不做任何處理。麻醉期間低濃度給氧(氧流量2 L·min-1),35℃干預(yù)。麻醉后,在相應(yīng)時(shí)間點(diǎn)斷頭提取海馬組織的總mRNA。

1.3 實(shí)時(shí)熒光定量PCR 用Trizol試劑盒提取各組海馬組織的總RNA,按實(shí)時(shí)定量RT-PCR試劑盒說(shuō)明書(shū)取500 ng總RNA逆轉(zhuǎn)錄合成cDNA,然后用熒光標(biāo)記物SYBR GreenI在ABI熒光定量PCR儀上進(jìn)行PCR反應(yīng)。在同一反應(yīng)體系內(nèi)對(duì)MEF2、Arc、synGAP I、synapsin I mRNA和內(nèi)參照β-actin mRNA進(jìn)行擴(kuò)增。PCR引物為:MEF2(NM_001014035.1)上游引物為5′-ACG GCA CAC AGA GCA CC TTG-3′,下游引物為5′-CTA GGT TTC TGC AGG CTA ATG TGGA-3′, PCR產(chǎn)物片斷為133 bp；Arc(ID:54323)上游引物為5′-GCA CAT AAA CCA TGA CCC ATA CT-3′,下游引物為5′-GCT GGA TAT TGA AGG CTT GG-3′,PCR產(chǎn)物片斷為111 bp；synGAP I(NM_001113409.1)上游引物為5′-TGT CCG CTG ACA TCG AGA GTG-3′,下游引物為5′-AGC TTC CGG TTG GAC ATG TGT AG-3′, PCR產(chǎn)物片斷為157 bp；synapsin I(NM_019133)上游引物為5′-TCT GA CCA ATG CCT TCA ACC TTC-3′,下游引物為5′-CTG CGG ATG GTC TCA GCT TTC-3′, PCR產(chǎn)物片斷為87 bp。β-actin(NM_031144)上游引物為5′-TGA CAG GAT GCA GAA GGA GA-3′下游引物為5′-TAG AGC CAC CAA TCC ACA CA-3′,PCR產(chǎn)物片斷為104 bp。擴(kuò)增參數(shù):94℃變性5 s,60℃退火及延伸30 s,共反應(yīng)40個(gè)循環(huán),分析融解曲線。

2 結(jié)果

2.1 氯胺酮對(duì)發(fā)育期大鼠海馬MEF2信號(hào)通路的影響 發(fā)育期大鼠海馬MEF2 mRNA在氯胺酮麻醉2 h時(shí)表達(dá)開(kāi)始下調(diào)(t=3.83,P＜0.05),4 h時(shí)表達(dá)最低(t=3.07,P＜0.05)；Arc mRNA(t=3.58,P＜0.05)和synGAP I mRNA(t=3.52,P＜0.05)在氯胺酮處理6 h時(shí)表達(dá)最低(P＜0.05)。于麻醉后24 h時(shí)3種基因mRNA水平表達(dá)恢復(fù)正常。見(jiàn)表1。故氯胺酮可短暫下調(diào)發(fā)育期大鼠海馬MEF2信號(hào)通路。

2.2 氯胺酮對(duì)發(fā)育期大鼠海馬synapsin I mRNA表達(dá)的影響 synapsin I mRNA麻醉4 h時(shí)表達(dá)開(kāi)始上調(diào)(t=-3.28,P＜0.05),6 h時(shí)表達(dá)達(dá)到高峰(t=-2.77,P＜0.05)。于麻醉后24 h時(shí)基因synapsin I mRNA水平表達(dá)恢復(fù)正常。見(jiàn)表2。故氯胺酮可上調(diào)發(fā)育期大鼠海馬synapsin I mRNA表達(dá)。

3 討論

氯胺酮具有鎮(zhèn)痛作用顯著而呼吸抑制作用較輕的特點(diǎn),多用于小兒麻醉。研究證實(shí)氯胺酮作為N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid,NMDA)受體非選擇性阻斷藥,可引起發(fā)育期神經(jīng)系統(tǒng)退行性變。其機(jī)制可能是氯胺酮阻斷NMDA受體,可抑制細(xì)胞外鈣離子內(nèi)流,抑制鈣/鈣調(diào)蛋白(Ca2+/CaM)的依賴(lài)信號(hào)途徑,從而抑制環(huán)磷酸腺苷反應(yīng)元件結(jié)合蛋白(cAMP-response element binding protein,CREB)和MEF2多種信號(hào)通路轉(zhuǎn)錄[5]。突觸形成期為大腦快速發(fā)育期,而NMDA受體活性是神經(jīng)系統(tǒng)在發(fā)育期神經(jīng)元生長(zhǎng)及突觸重塑所必需。由于突觸形成期神經(jīng)元對(duì)各種麻醉藥最為敏感,發(fā)育期大鼠神經(jīng)元發(fā)育高峰期為出生后第1～7 d,一般認(rèn)為第5天是神經(jīng)元發(fā)育的高峰期并開(kāi)始形成功能性突觸連接[6-7]。因此筆者采用5 d齡乳鼠來(lái)源的海馬神經(jīng)元,用作發(fā)育期海馬神經(jīng)元來(lái)探討氯胺酮對(duì)突觸生長(zhǎng)和突觸重塑的影響。

表1 兩組大鼠各時(shí)間點(diǎn)海馬MEF2 mRNA、synGAP I mRNA、Arc mRNA表達(dá)水平的比較Tab.1 Comparison of the mRNA expression of MEF2,synGAP I and Arc in hippocampal neurons between two groups at each time point n=3,±s

表1 兩組大鼠各時(shí)間點(diǎn)海馬MEF2 mRNA、synGAP I mRNA、Arc mRNA表達(dá)水平的比較Tab.1 Comparison of the mRNA expression of MEF2,synGAP I and Arc in hippocampal neurons between two groups at each time point n=3,±s

與C組同時(shí)間點(diǎn)比較,*1P＜0.05Compared with C group at same time point,*1P＜0.05

組別與時(shí)間MEF2 mRNAsynGAP I mRNAArc mRNA組別與時(shí)間MEF2 mRNAsynGAP I mRNAArc mRNA T組C組2 h0.69±0.09*10.76±0.07*10.70±0.14*12 h1.01±0.101.03±0.050.99±0.09 4 h0.52±0.13*10.61±0.12*10.55±0.21*14 h1.01±0.121.03±0.091.02±0.17 6 h0.66±0.14*10.57±0.17*10.48±0.22*16 h0.99±0.130.98±0.100.98±0.10 24 h1.03±0.041.03±0.190.96±0.08 24 h1.02±0.091.01±0.110.97±0.06

表2 兩組大鼠各時(shí)間點(diǎn)海馬synapsin I mRNA表達(dá)水平的比較Tab.2 Comparison of synapsin I mRNA expression in hippocampal neurons between two groups at each time point n=3,±s

表2 兩組大鼠各時(shí)間點(diǎn)海馬synapsin I mRNA表達(dá)水平的比較Tab.2 Comparison of synapsin I mRNA expression in hippocampal neurons between two groups at each time point n=3,±s

與C組同時(shí)間點(diǎn)比較,*1P＜0.05Compared with C group at same time point,*1P＜0.05

組別2 h4 h6 h24 h T組1.29±0.341.90±0.47*12.46±0.91*11.18±0.57 C組1.06±0.210.96±0.140.99±0.140.98±0.05

MEF2是一種特定的轉(zhuǎn)錄因子,因其涉及基因調(diào)節(jié)不同環(huán)節(jié)及其控制多種基因表達(dá),正日益受到高度重視[3-4]。目前發(fā)現(xiàn)MEF2在發(fā)育期神經(jīng)元中是調(diào)節(jié)突觸生長(zhǎng)和突觸重塑的重要信號(hào)通路。在發(fā)育期中樞神經(jīng)系統(tǒng),MEF2的激活負(fù)調(diào)控興奮性突觸密度。MEF2激活依賴(lài)于細(xì)胞外Ca2+內(nèi)流。鈣離子通過(guò)NMDA和電壓依賴(lài)性鈣通道(voltage-dependentcalcium channels,VDCCs)內(nèi)流,激活鈣/鈣調(diào)蛋白(Ca2+/CaM)的依賴(lài)信號(hào)途徑。然后刺激鈣調(diào)磷酸酶(也稱(chēng)為蛋白磷酸酶2B)去磷酸化MEF2的蛋白質(zhì)上一些磷酸化位點(diǎn),包括對(duì)MEF2A的和MEF2D(分別抑制Ser408和Ser444),以促進(jìn)MEF2的活化。當(dāng)MEF2被激活,促進(jìn)了其一系列下游基因轉(zhuǎn)錄,包括Arc和synGAP[8]。研究證實(shí)Arc是調(diào)控發(fā)育期海馬神經(jīng)元的突觸數(shù)目重要基因[3-4]。因此本研究旨在探討氯胺酮對(duì)MEF2信號(hào)通路及其下游基因的表達(dá)的影響。

Synapsin I是特異性位于軸突末梢的突觸前膜上,是一種重要的膜標(biāo)記蛋白。Synapsin I在發(fā)育期神經(jīng)元生長(zhǎng)及突觸重塑方面起著非常重要的作用,它參與乙酰膽堿、谷氨酸等神經(jīng)遞質(zhì)的釋放過(guò)程。在發(fā)育期神經(jīng)元軸突生長(zhǎng)過(guò)程中synapsin I調(diào)控著軸突分支數(shù)目。有研究認(rèn)為,synapsin I一方面通過(guò)對(duì)突觸結(jié)構(gòu)的影響,另一方面通過(guò)其磷酸化作用調(diào)節(jié)神經(jīng)遞質(zhì)的釋放,從而在突觸可塑性中起一定作用[1-2]。筆者研究證實(shí)氯胺酮可以上調(diào)發(fā)育期海馬神經(jīng)元synapsin I表達(dá),這說(shuō)明氯胺酮可能增加軸突分支的數(shù)目有關(guān)。

本研究結(jié)果表明,發(fā)育期大鼠海馬MEF2 mRNA、synGAP I mRNA和Arc mRNA在氯胺酮麻醉處理后短暫表達(dá)下調(diào),說(shuō)明氯胺酮能夠抑制MEF2信號(hào)通路。氯胺酮麻醉處理后發(fā)育期大鼠海馬synapsin I表達(dá)都短暫上調(diào),說(shuō)明氯胺酮增強(qiáng)對(duì)synapsin I表達(dá)。

綜上所述,氯胺酮可短暫下調(diào)MEF2信號(hào)通路(MEF2 mRNA、synGAP I mRNA和Arc mRNA)的表達(dá),且上調(diào)海馬突觸形成相關(guān)基因synapsin I表達(dá)。在氯胺酮作用下MEF2信號(hào)通路對(duì)synapsin I表達(dá)的調(diào)節(jié)關(guān)系,還需進(jìn)一步實(shí)驗(yàn)證實(shí)。

[1] MULLER H K,WEGENER G,LIEBENBERG N,et al.Ketamine regulates the presynaptic release machinery in the hippocampus[J].J Psychiatr Res,2013,47(7):892-899.

[2] SINNER B,FRIEDRICH O,ZINK W,et al.The toxic effects of s(+)-ketamine on differentiating neuronsin vitroas a consequence of suppressed neuronal Ca2+oscillations[J]. Anesth Analg,2011,113(5):1161-1169.

[3] 趙以林,羅愛(ài)林,金小高,等.異氟醚麻醉對(duì)新生大鼠海馬激活肌細(xì)胞增強(qiáng)因子2信號(hào)通路的影響[J].中華麻醉學(xué)雜志,2011,31(6):714-716.

[4] FLAVELL S W,COWAN C W,KIM T K,et al.Activity-dependent regulation of MEF2 transcription factors suppresses excitatory synapse number[J].Science,2006,311(5763): 1008-1012.

[5] HUANG L,LIU Y,ZHANG P,et al.In vitrodose-dependent inhibition of the intracellular spontaneous calcium oscillations in developing hippocampal neurons by ketamine[J].PLoS One,2013,8(3):e59804.

[6] ZHAO Y L,XIANG Q,SHI Q Y,et al.GABAergic excitotoxicity injury of the immature hippocampal pyramidal neurons'exposure to isoflurane[J].Anesth Analg,2011,113 (5):1152-1160.

[7] ZHAO Y,JIN X,WANG J,et al.Isoflurane enhances the expression of cytochrome C by facilitation of NMDA receptor in developing rat hippocampal neuronsin vitro[J].J Huazhong Univ Sci Technolog Med Sci,2011,31(6):779-783.

[8] SMITH J A,KOHN T A,CHETTY A K,et al.CaMK activation during exercise is required for histone hyperacetylation and MEF2A binding at the MEF2 site on the Glut4 gene[J]. Am J Physiol Endocrinol Metab,2008,295(3):698-704.

DOI 10.3870/yydb.2014.04.002

Effects of Ktamine on Transcriptional Factor MEF2 Signaling Pathway and Expression of Synaptogenesis Synapsin I of Rat Developing Hippocampal Neurons in vivo

LUO Xiao-xiao,ZHAO Yi-lin,LI Shi-yong,TAN Lei,WANG Jin-tao
(The Second Clinical College of Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)

Objective To investigate the time-dependent effects of ketamine on myocyte enhancer factor 2(MEF2) signaling pathway and expression of synaptogenesis synapsin I of rat developing hippocampal neuronsin vivo.MethodsForty 5-day-old rats were randomly divided into 4 treatment groups receiving ketamine(20 mg·kg-1)single injection and a control group receiving no treatment.Pups were killed by decapitation to extract total RNA from hippocampal neurons in the 4 treatment groups at 2,4,6,24 h,respectively,and from the control group.Real-time-PCR was used to detect the expression of the MEF2 mRNA、synGAP I mRNA and Arc mRNA and synapsin I mRNA after intervention.ResultsCompared with the control group, there are significant decreases in the expression of MEF2 mRNA,synGAP I mRNA,Arc mRNA and increase in synapsin I mRNA of the neurons from ketamine treatment groups(P＜0.05).ConclusionThe mechanism of ketamine-induced decrease in expression of MEF2 signaling pathway and increase in the expression of synapsin I may be related to decreased expression of Arc signaling pathway,which in turn regulates the expression of snapsin I in the developing hippocampal neurons.

Ketamine;Hippocampus;Developing neuron;Myocyte enhancer factor 2;Synapsin I

R971.2;R965

A

1004-0781(2014)04-0419-03

2013-05-02

2013-10-25

*國(guó)家自然科學(xué)基金資助項(xiàng)目(81200880)

羅蕭蕭(1992-),女,湖北武漢人,學(xué)士,研究方向:麻醉藥理。電話(huà):(0)13507122565,E-mail：asd2007asd@126.com。

趙以林(1982-),男,山東日照人,醫(yī)師,博士,研究方向:麻醉毒理。電話(huà):(0)13871469616,E-mail：yilinzhao001@163.com。