楊展能，姜靚婧，顧仕紅，陳磊，黎虹薇，朱新建

(東南大學 醫(yī)學院，江蘇 南京 210009)

·論 著·

NMDA受體在匹羅卡品癲癇小鼠海馬星形膠質(zhì)細胞活化中的作用

楊展能，姜靚婧，顧仕紅，陳磊，黎虹薇，朱新建

(東南大學 醫(yī)學院，江蘇 南京 210009)

目的：探討在匹羅卡品誘導的小鼠癲癇持續(xù)狀態(tài)(SE)模型中，N- 甲基- D- 天門冬氨酸(NMDA)受體在海馬星形膠質(zhì)細胞增生中的作用及其可能的分子機制。方法：將32只6周齡雄性C57/BL6小鼠隨機分為對照組、SE組、SE后注射NMDA受體拮抗劑MK- 801組和單純注射MK- 801組，每組8只。腹腔注射300 mg·kg-1匹羅卡品建立SE模型。免疫組化方法檢測各組小鼠海馬腦區(qū)星形膠質(zhì)細胞的形態(tài)差異，同時利用western Blot方法檢測海馬腦區(qū)神經(jīng)膠質(zhì)纖維酸性蛋白(GFAP)的表達和轉(zhuǎn)錄因子cAMP反應元件結(jié)合蛋白(CREB)的磷酸化水平。結(jié)果：SE組小鼠海馬星形膠質(zhì)細胞出現(xiàn)顯著活化現(xiàn)象，免疫組化結(jié)果顯示SE組小鼠海馬GFAP免疫反應強度顯著高于對照組(P<0.01)，而在SE后給予MK- 801阻斷NMDA受體則顯著抑制了SE誘導的海馬星形膠質(zhì)細胞活化。與免疫組化結(jié)果一致，Western Blot結(jié)果顯示SE組小鼠海馬GFAP蛋白表達水平顯著高于對照組(P<0.05)，而在SE后給予MK- 801阻斷NMDA受體則顯著抑制了SE誘導的海馬GFAP蛋白表達水平的升高。同時我們的研究結(jié)果發(fā)現(xiàn)，SE組小鼠海馬CREB蛋白磷酸化水平顯著高于對照組，而阻斷NMDA受體則顯著抑制了SE誘導的海馬CREB蛋白磷酸化。結(jié)論：匹羅卡品癲癇小鼠持續(xù)狀態(tài)7 d后海馬星形膠質(zhì)細胞被顯著活化，而這一活化過程依賴于NMDA受體的激活。同時，海馬星形膠質(zhì)細胞活化的過程伴隨CREB的磷酸化，提示海馬CREB磷酸化參與了NMDA受體介導的匹羅卡品癲癇小鼠星形膠質(zhì)細胞活化過程。

N- 甲基- D- 天門冬氨酸受體； 匹羅卡品； 癲癇； 海馬； 星形膠質(zhì)細胞活化； 小鼠

N- 甲基- D- 天門冬氨酸(N- methy- D- asparate, NMDA)受體一直是基礎(chǔ)和臨床研究的焦點。生理狀態(tài)下，NMDA受體在突觸傳遞、突觸可塑性以及學習記憶等過程中起著重要作用[1- 4]，然而當其被過度激活后所致的興奮性毒性可導致某些神經(jīng)系統(tǒng)疾病[5- 7]。研究發(fā)現(xiàn)，癲癇發(fā)作時神經(jīng)元興奮性增強使興奮性谷氨酸遞質(zhì)大量釋放，大量的谷氨酸遞質(zhì)過度激活神經(jīng)元膜上的NMDA受體引起Ca2+內(nèi)流，導致細胞內(nèi)信號過度激活和神經(jīng)元興奮性異常增高，并最終引起中樞神經(jīng)系統(tǒng)內(nèi)某些特定神經(jīng)環(huán)路結(jié)構(gòu)和功能的異常改變從而引發(fā)癲癇疾病[8- 9]。癲癇發(fā)作引起的腦內(nèi)特殊的病理改變，尤其是海馬結(jié)構(gòu)的損傷，如海馬神經(jīng)元丟失(neuron loss)、星形膠質(zhì)細胞活化(astrocytes activation)、神經(jīng)元再生(neurogenesis)、苔蘚纖維發(fā)芽(mossy fiber sprouting)、炎癥反應(inflammation)等[10- 15]，而這些海馬結(jié)構(gòu)損傷是異常興奮性突觸環(huán)路形成的重要原因，可促進形成難治性癲癇疾病[16- 17]。本研究通過建立匹羅卡品(pilocarpine)小鼠癲癇持續(xù)狀態(tài)(status epilepticus, SE)模型，探討小鼠SE后海馬星形膠質(zhì)細胞活化的情況并揭示NMDA受體在SE后海馬星形膠質(zhì)細胞活化中的作用及其可能的分子機制。

1 材料和方法

1.1 動物和分組

32只6周齡雄性成年C57/BL6小鼠，體重18～22 g,由揚州大學比較實驗動物中心提供。小鼠于室溫18～25 ℃、人工12 h晝夜循環(huán)照明條件下飼養(yǎng)，自由攝食與飲水。小鼠隨機分為對照組、SE組、SE后注射NMDA受體拮抗劑MK- 801組和單純注射MK- 801組，每組8只。

1.2 主要試劑和儀器

匹羅卡品、東莨菪堿、MK- 801(Sigma Aldrich)，神經(jīng)膠質(zhì)纖維酸性蛋白(GFAP)抗體(武漢博士得生物)，磷酸化cAMP反應元件結(jié)合蛋白(pCREB)、CREB抗體(Millipore, Billerica, MA, USA)，β- actin抗體(華安生物)，辣根過氧化物酶偶聯(lián)羊抗兔和羊抗小鼠二抗(武漢博士得生物)，F(xiàn)ITC偶聯(lián)山羊抗小鼠熒光二抗(世紀康為)，RIPA蛋白裂解液(碧云天)，BCA蛋白定量分析試劑盒(Pierce，Rockford, IL, USA)，增強型ECL發(fā)光液(Super Signal West Pico Trial Kit，Pierce)，5%脫脂奶粉(BD- Difco，USA)，DAPI- 抗猝滅封片劑(SouthernBiotech, Birmingham, AL, USA)，硝酸纖維素膜(Amersham, LittleChalfont, UK)，冷凍切片機(Leica Microsystems, Wetzlar, Germany)，激光共聚焦顯微鏡(Olympus LSM- GB200, Japan)，Western Blot化學發(fā)光圖像分析系統(tǒng)(DNR Bio- imaging Systems, Jerusalem, Israel)，蛋白電泳轉(zhuǎn)移設備(Bio- Rad, Hercules, CA, USA)，Immage J軟件(NIH, Bethesda, MD, USA)。

1.3 動物處理

SE組、SE后注射NMDA受體拮抗劑MK- 801組小鼠建立癲癇模型：給予匹羅卡品300 mg·kg-1腹腔注射誘導SE，注射匹羅卡品前30 min給予1 mg·kg-1東莨菪堿腹腔注射以阻斷外周膽堿能作用，觀察動物癲癇發(fā)作，根據(jù)Racine標準[18]判定癲癇發(fā)作級別。0級:無抽搐發(fā)作；1級:耳、面部抽搐；2級:肌陣攣，但無直立位；3級:肌陣攣，伴直立位；4級:全身強直陣攣發(fā)作；5級:強直陣攣發(fā)作并失去體位控制。達到癲癇發(fā)作3～5級的存活小鼠入選。SE后注射NMDA受體拮抗劑MK- 801組小鼠在SE結(jié)束后腹腔注射1 mg·kg-1MK- 801(連續(xù)注射7 d，每天1次)；對照組腹腔注射等量用于配置匹羅卡品的生理鹽水；單純注射MK- 801組小鼠腹腔注射1 mg·kg-1MK- 801(連續(xù)注射7 d，每天1次)。

1.4 Western Blot檢測

取小鼠雙側(cè)海馬置于RIPA蛋白裂解液中，冰浴上迅速勻漿，勻漿完畢后將樣品置于冰浴上裂解15 min，2 000 r·min-1離心10 min，小心吸取上清即為蛋白提取液，-80 ℃保存。Brad法測定蛋白濃度，根據(jù)所測定的的蛋白濃度將所有蛋白樣品調(diào)至等濃度，然后于待測蛋白樣品中加入0.2體積的6×蛋白加樣緩沖液 [300 mmo1·L-1Tris(pH 6.8)、600 mmo1·L-1DTT、12% SDS、0.6%澳酚藍、60%甘油]100 ℃煮沸3 min， 12 000 r·min-1離心10 min，取上清至-20 ℃保存待分析。灌制12%的SDS- PAGE膠，恒壓100 V電泳分離海馬組織蛋白樣品。電泳結(jié)束后，組織蛋白樣品在轉(zhuǎn)移液(9 mmol·L-1甘氨酸、48 mmo1·L-1Tris堿、0.037% SDS、20%甲醇)中300 mA恒流轉(zhuǎn)移2.5 h至硝酸纖維素膜。5%脫脂奶粉封閉1 h，TBST洗膜10 min×3次，小鼠GFAP抗體(1∶2 000)和兔p-CREB/CREB抗體(1∶2 000)孵育過夜。次日，棄去一抗，TBST洗膜10min×3次，辣根過氧化物酶標記的羊抗小鼠(1∶5 000)和羊抗兔二抗(1∶10 000)孵育2h，TBST洗膜10min×3次，末次洗膜后迅速以ECL發(fā)光液試劑A液和B液各500μl混合后于膜上反應0.5～1.0min，WesternBlot化學發(fā)光系統(tǒng)采集信號，ImmageJ軟件進行數(shù)據(jù)分析。

1.5 免疫組化檢測

腹腔注射過量的烏拉坦麻醉小鼠，經(jīng)左心室依次灌入200ml生理鹽水和4%多聚甲醛(PA)200ml，小心將鼠腦取出并將其置于4%多聚甲醛中固定過夜。取出固定后的鼠腦，選擇海馬部位，用震蕩切片機進行冠狀連續(xù)切片，腦片厚度為25μm，每隔200μm取腦片1張，每只小鼠取12張腦片，收集于PBS(pH7.4)中。切片于50%甲酰胺(formamide,Sigma)/2×SSC中65 ℃加熱2h,PBS(pH=7.4)洗片5min×3次，2mol·L-1HCl37 ℃孵育30min，0.1mol·L-1硼酸(pH8.5)洗片10min，PBS洗片1次，3%H2O2(PBS稀釋)室溫孵育30min,PBS洗片5min×3次，封閉血清室溫孵育60min，棄去封閉血清但不洗片，加入小鼠GFAP抗體(1∶200，稀釋于山羊血清)4 ℃孵育過夜。次日棄去一抗并用PBS洗片5min×3次，加入FITC偶聯(lián)山羊抗小鼠熒光二抗(1∶200)室溫孵育2h，棄去二抗并用PBS洗片5min×3次。染好的腦片爬片，晾干后用中DAPI抗猝滅封片劑封片。普通熒光顯微鏡下觀察海馬GFAP陽性細胞，激光共聚焦顯微鏡拍照。

1.6 統(tǒng)計學處理

采用SigmaPlot(11.0)軟件進行統(tǒng)計學分析，數(shù)據(jù)以平均數(shù)±標準誤表示，組間多樣本均數(shù)比較采用ANOVA方差分析。P<0.05為差異具有統(tǒng)計學意義。

2 結(jié) 果

2.1 匹羅卡品癲癇小鼠持續(xù)狀態(tài)行為學觀察

小鼠腹腔注射匹羅卡品(300 mg·kg-1)10～15 min后出現(xiàn)運動減少、凝視不動、咀嚼、流涎、須動、濕狗樣抖動、耳面部抽搐和平衡失調(diào)等行為學變化。隨后進一步出現(xiàn)前肢陣攣及(或)肢體強直陣攣伴直立、跌倒或翻轉(zhuǎn)等邊緣系統(tǒng)運動性發(fā)作，呈持續(xù)性3～5級發(fā)作即為SE。

2.2 匹羅卡品癲癇小鼠海馬星形膠質(zhì)細胞活化情況

給予匹羅卡品300 mg·kg-1腹腔注射誘導SE，注射匹羅卡品前30 min給予1 mg·kg-1東莨菪堿腹腔注射以阻斷外周膽堿能作用，觀察動物癲癇發(fā)作，根據(jù)Racine標準[18]判定癲癇發(fā)作級別。癲癇發(fā)作未達3級、超過5級或死亡者均剔除。SE后7 d分別利用免疫組化方法和Western Blot方法檢測海馬GFAP表達情況(圖1)。免疫組化結(jié)果顯示，匹羅卡品癲癇小鼠海馬GFAP標記的陽性細胞數(shù)量較對照組顯著增加，并且出現(xiàn)細胞分支增多的現(xiàn)象(圖2A)。數(shù)據(jù)統(tǒng)計分析顯示，匹羅卡品癲癇小鼠的GFAP免疫熒光強度顯著高于對照組小鼠(圖2B)。Western Blot結(jié)果顯示匹羅卡品癲癇小鼠海馬GFAP的蛋白水平較對照組有顯著升高(圖3A、B)，提示匹羅卡品癲癇小鼠海馬星形膠質(zhì)細胞被顯著活化。

2.3 匹羅卡品癲癇小鼠海馬星形膠質(zhì)細胞活化與NMDA受體激活的關(guān)系

為了進一步探索匹羅卡品癲癇小鼠海馬星形膠質(zhì)細胞被顯著活化是否依賴于NMDA受體的激活，我們在SE后給小鼠持續(xù)腹腔注射NMDA受體拮抗劑MK- 801后分別利用免疫熒光和Western Blot檢測海馬GFAP表達情況。免疫組織化學結(jié)果顯示，SE后注射NMDA受體拮抗劑MK- 801顯著抑制了誘導的GFAP陽性細胞數(shù)量的增加，而單獨給予小鼠MK- 801后GFAP陽性細胞數(shù)量與對照組比較未見顯著差異。與免疫組織化學結(jié)果一致，Western Blot結(jié)果顯示SE后注射NMDA受體拮抗劑MK- 801顯著抑制了癲癇持續(xù)狀態(tài)誘導的GFAP蛋白表達的增加，而單獨給予MK- 801后GFAP蛋白表達水平較對照組比較未見顯著差異。以上結(jié)果證實，匹羅卡品癲癇小鼠海馬星形膠質(zhì)細胞被顯著活化依賴于NMDA受體的激活。

圖1 小鼠匹羅卡品癲癇持續(xù)狀態(tài)模型的建立

Fig 1 Schematic representation of pilocarpine- induced SE mice model

A.對照組、SE組、SE后注射NMDA受體拮抗劑MK- 801組和單純注射MK- 801組小鼠海馬GFAP免疫熒光標記；B.各組小鼠海馬GFAP免疫熒光強度定量分析結(jié)果(n=5)

aP<0.05，bP<0.01，比例尺=100 μm

A.Representative immunofluorescence images of GFAP-staining in the hippocampus of Control, SE, SE+MK- 801 and MK- 801- treated mice;B.Bar graph showed the quantification of GFAP immunostaining intensity in the hippocampus of Control, SE, SE+MK- 801 and MK- 801- treated mice(n=5)

aP<0.05，bP<0.01, scale bar=100 μm

圖2 免疫組化檢測匹羅卡品癲癇小鼠海馬星形膠質(zhì)細胞活化

Fig 2 Hippocampal astrocytes were activated in SE mice

A.對照組、SE組、SE后注射NMDA受體拮抗劑MK- 801組和單純注射MK- 801組小鼠海馬GFAP Western Blot檢測結(jié)果；B.上述各組小鼠海馬GFAP Western Blot定量分析結(jié)果(n=5)

aP<0.05，bP<0.01

A.Western Blot assays of GFAP expression in the hippocampus of Control, SE, SE+MK- 801 and MK- 801- treated mice; B.Bar graph showed the quantification of hippocampal GFAP protein levels, which were represented as the intensity ratio of GFAP to β- actin in the hippocampus of Control, SE, SE+MK- 801 and MK- 801- treated mice(n=5)

aP<0.05，bP<0.01

圖3 Western Blot檢測匹羅卡品癲癇小鼠海馬星形膠質(zhì)細胞活化

Fig 3 Western Blot assays of astrocytes activation in the hippocampus of SE mice

2.4 匹羅卡品癲癇小鼠海馬CREB磷酸化水平與NMDA受體激活的關(guān)系

CREB作為一種刺激誘導型轉(zhuǎn)錄因子廣泛分布于中樞神經(jīng)系統(tǒng)，有研究發(fā)現(xiàn)CREB與抑郁癥密切相關(guān)[19]，我們的早期研究結(jié)果顯示，SE發(fā)生后皮層和海馬中的pCREB水平顯著提高，提示CREB在癲癇發(fā)生的病理過程中具有重要作用。在本研究中，我們利用匹羅卡品腹腔注射誘導SE 7 d后，利用Western Blot方法檢測海馬總的CREB以及pCREB水平，并計算分析海馬CREB磷酸化水平。我們的研究結(jié)果顯示，匹羅卡品癲癇小鼠海馬CREB的磷酸化水平較對照組有顯著升高，SE后注射NMDA受體拮抗劑MK- 801則顯著抑制了SE誘導的CREB磷酸化水平的增加，而單獨給予小鼠MK- 801后CREB的蛋白磷酸化水平與對照組比較未見顯著差異(圖4)。以上結(jié)果說明匹羅卡品誘導的SE誘導了轉(zhuǎn)錄因子CREB磷酸化，且這一過程依賴于NMDA受體的激活。

3 討 論

神經(jīng)膠質(zhì)細胞是廣泛分布于中樞神經(jīng)系統(tǒng)內(nèi)的支持細胞，具有支持、滋養(yǎng)神經(jīng)元的作用。其中星形膠質(zhì)細胞具有支持和引導神經(jīng)元遷移，并為神經(jīng)元運輸營養(yǎng)物質(zhì)和排除代謝產(chǎn)物[20- 21]的作用。新近的研究表明，星形膠質(zhì)細胞在中樞神經(jīng)系統(tǒng)除了具有支持和營養(yǎng)作用，同時還參與神經(jīng)元的突觸傳遞活動[22]。星形膠質(zhì)細胞與神經(jīng)元細胞膜緊緊相鄰，而且表面分布有大量的受體、轉(zhuǎn)運蛋白和離子通道，這為星形膠質(zhì)細胞和神經(jīng)元之間的交換作用創(chuàng)造了必要條件。大量的研究表明，在一些病理狀態(tài)下星形膠質(zhì)細胞從靜止態(tài)向活化態(tài)轉(zhuǎn)變，其結(jié)構(gòu)和功能均發(fā)生改變，并通過神經(jīng)遞質(zhì)和離子通道進一步調(diào)控神經(jīng)元的活動。

GFAP是星形膠質(zhì)細胞的骨架蛋白，被公認為是星形膠質(zhì)細胞的特征標志物。我們先前的研究發(fā)現(xiàn)戊四唑(pentylenetetrazole，PTZ)誘導的點燃癲癇模型中海馬組織發(fā)生星形膠質(zhì)細胞活化的現(xiàn)象，表現(xiàn)為GFAP標記的星形膠質(zhì)細胞數(shù)量增加、分支增多[23]。本研究在匹羅卡品誘導的SE模型小鼠中觀察到海馬GFAP標記的星形膠質(zhì)細胞數(shù)量顯著增加，并且GFAP陽性細胞分支增多的現(xiàn)象，Western Blot結(jié)果顯示匹羅卡品癲癇小鼠海馬GFAP的蛋白水平較對照組有顯著升高，提示匹羅卡品SE小鼠海馬星形膠質(zhì)細胞被顯著活化。

盡管目前已知星形膠質(zhì)細胞活化是癲癇疾病中的一個典型的病理性損傷，然而在癲癇疾病中介導星形膠質(zhì)細胞活化的確切的細胞分子機制仍然不是很清楚。NMDA受體在癲癇疾病的病理生理學過程具有重要的作用。我們先前的研究發(fā)現(xiàn)NMDA受體在PTZ誘導的點燃癲癇模型中被激活，并在海馬星形膠質(zhì)細胞活化和氧化應激過程中發(fā)揮了重要作用[23]。本研究在SE后給予MK- 801阻斷NMDA受體，發(fā)現(xiàn)其顯著抑制了SE誘導的海馬GFAP蛋白表達水平的升高，提示匹羅卡品癲癇小鼠海馬星形膠質(zhì)細胞活化依賴于NMDA受體的激活。

A.對照組、SE組、SE后注射NMDA受體拮抗劑MK- 801組和單純注射MK- 801組小鼠海馬CREB、pCREB Western Blot檢測結(jié)果；B.上述各組小鼠海馬CREB磷酸化(以pCREB/CREB表示)定量分析結(jié)果(n=5)

aP<0.05，bP<0.01

A.Western Blot assays of phosphorylated CREB(pCREB) and CREB expression in the hippocampus of Control, SE, SE+MK- 801 and MK- 801- treated mice; B.Bar graph showed the quantification of hippocampal CREB phosphorylation level, which were represented as the intensity ratio of pCREB to CREB in the hippocampus of Control, SE, SE+MK- 801 and MK- 801- treated mice(n=5)

aP<0.05，bP<0.01

圖4 Western Blot檢測匹羅卡品癲癇小鼠海馬CREB磷酸化水平

Fig 4 Western Blot assays of CREB phosphorylation in the hippocampus of SE mice

CREB作為一種刺激誘導型轉(zhuǎn)錄因子廣泛分布于中樞神經(jīng)系統(tǒng)[24]，在神經(jīng)元中大量的細胞外刺激因子通過磷酸化激活CREB啟動CREB依賴的下游基因的轉(zhuǎn)錄[25- 27]，從而參與許多生理及病理機制的調(diào)節(jié)。生理狀態(tài)下，CREB在中樞神經(jīng)系統(tǒng)中的主要功能包括調(diào)控神經(jīng)元的增殖、分化、生長的過程[24,28]以及調(diào)節(jié)學習、記憶的功能和突觸可塑性[29- 30]。新近的研究發(fā)現(xiàn)，CREB轉(zhuǎn)錄因子介導的轉(zhuǎn)錄過程參與癲癇發(fā)生過程并起到重要作用[31- 32]。有研究發(fā)現(xiàn)，SE發(fā)生后，皮層和海馬中的pCREB水平顯著提高[33]。CREB不僅在癲癇發(fā)生的過程中被激活，它還主動參與調(diào)控癲癇的發(fā)生過程。我們先前的研究發(fā)現(xiàn)通過遺傳學手段抑制CREB顯著遏制了匹羅卡品誘導的持續(xù)癲癇以及自發(fā)性癲癇的發(fā)生[34- 35]。本研究中我們在匹羅卡品誘導的SE小鼠中觀察到CREB磷酸化水平顯著增高，且這一過程依賴于NMDA受體的激活，提示CREB可能參與了海馬星形膠質(zhì)細胞活化的過程。SE后NMDA受體被激活進而導致大量的Ca2+內(nèi)流，Ca2+作為第二信使激活了其下游一系列的信號通路參與癲癇發(fā)生的病理過程調(diào)控，而CREB作為一種誘導型的轉(zhuǎn)錄因子，在癲癇發(fā)作后可被上游的分子信號所激活。由此我們推測，SE后被激活的NMDA受體通過CREB轉(zhuǎn)錄因子調(diào)控了星形膠質(zhì)細胞的活化過程。

[1] LEE I,KESNER R P.Differential contribution of NMDA receptors in hippocampal subregions to spatial working memory[J].Nat Neurosci,2002,5(2):162- 168.

[2] von ENGELHARDT J,DOGANCI B,JENSEN V,et al.Contribution of hippocampal and extra- hippocampal NR2B- containing NMDA receptors to performance on spatial learning tasks[J].Neuron,2008,60(5):846- 860.

[3] HEPP Y,SALLES A,CARBO- TANO M,et al.Surface expression of NMDA receptor changes during memory consolidation in the crab Neohelice granulata[J].Learn Mem,2016,23(8):427- 434.

[4] KUNZ P A,ROBERTS A C,PHILPOT B D.Presynaptic NMDA receptor mechanisms for enhancing spontaneous neurotransmitter release[J].J Neurosci,2013,33(18):7762- 7769.

[5] HARA M R,SNYDER S H.Cell signaling and neuronal death[J].Annu Rev Pharmacol Toxicol,2007,47:117- 141.

[6] STANIKA R I,PIVOVAROVA N B,BRANTNER C A,et al.Coupling diverse routes of calcium entry to mitochondrial dysfunction and glutamate excitotoxicity[J].Proc Natl Acad Sci U S A,2009,106(24):9854- 9859.

[7] OKAMOTO S,POULADI M A,TALANTOVA M,et al.Balance between synaptic versus extrasynaptic NMDA receptor activity influences inclusions and neurotoxicity of mutant huntingtin[J].Nat Med,2009,15(12):1407- 1413.

[8] MCNAMARA J O,HUANG Y Z,LEONARD A S.Molecular signaling mechanisms underlying epileptogenesis[J].Sci STKE,2006,2006(356):re12.

[9] GHASEMI M,SCHACHTER S C.The NMDA receptor complex as a therapeutic target in epilepsy:a review[J].Epilepsy Behav,2011,22(4):617- 640.

[10] BUCKMASTER P S,LEW F H.Rapamycin suppresses mossy fiber sprouting but not seizure frequency in a mouse model of temporal lobe epilepsy[J].J Neurosci,2011,31(6):2337- 2347.

[11] PEIXOTO- SANTOS J E,VELASCO T R,GALVIS- ALONSO O Y,et al.Temporal lobe epilepsy patients with severe hippocampal neuron loss but normal hippocampal volume:Extracellular matrix molecules are important for the maintenance of hippocampal volume[J].Epilepsia,2015,56(10):1562- 1570.

[12] GOUBRAN M,BERNHARDT B C,CANTOR- RIVERA D,et al.InvivoMRI signatures of hippocampal subfield pathology in intractable epilepsy[J].Hum Brain Mapp,2016,37(3):1103- 1119.

[13] CHO K O,LYBRAND Z R,ITO N,et al.Aberrant hippocampal neurogenesis contributes to epilepsy and associated cognitive decline[J].Nat Commun,2015,6(6606):1- 13.

[14] ALSHARAFI W A,XIAO B,ABUHAMED M M,et al.Correlation between IL- 10 and microRNA- 187 expression in epileptic rat hippocampus and patients with temporal lobe epilepsy[J].Front Cell Neurosci,2015,9(466):1- 9.

[15] GERSHEN L D,ZANOTTI- FREGONARA P,DUSTIN I H,et al.Neuroinflammation in temporal lobe epilepsy measured using positron emission tomographic imaging of translocator protein[J].JAMA Neurol,2015,72(8):882- 888.

[16] SCHARFMAN H E,SOLLAS A L,BERGER R E,et al.Electrophysiological evidence of monosynaptic excitatory transmission between granule cells after seizure- induced mossy fiber sprouting[J].J Neurophysiol,2003,90(4):2536- 2347.

[17] GABRIEL S,NJUNTING M,POMPER J K,et al.Stimulus and potassium- induced epileptiform activity in the human dentate gyrus from patients with and without hippocampal sclerosis[J].J Neurosci,2004,24(46):10416- 10430.

[18] RACINE R J.Modification of seizure activity by electrical stimulation.Ⅱ.Motor seizure[J].Electroencephalography and Clinical Neurophysiology,1972,32(3):281- 294.

[19] 董瓊,趙雪梅，袁天榮,等.CREB及其與抗抑郁癥相關(guān)性的研究進展[J].現(xiàn)代醫(yī)學,2014,42(6):710- 712.

[20] GARCIA- MARIN V,GARCIA- LOPEZ P,FREIRE M.Cajal’s contributions to glia research[J].Trends Neurosci,2007,30(9):479- 487.

[21] VERKHRATSKY A.Patching the glia reveals the functional organisation of the brain[J].Pflugers Arch,2006,453(3):411- 420.

[22] HALASSA M M,FELLIN T,HAYDON P G.The tripartite synapse:roles for gliotransmission in health and disease[J].Trends in Molecular Medicine,2007,13(2):54- 63.

[23] ZHU X,DONG J,SHEN K,et al.NMDA receptor NR2B subunits contribute to PTZ- kindling- induced hippocampal astrocytosis and oxidative stress[J].Brain Research Bulletin,2015,114:70- 78.

[24] LONZE B E,GINTY D D.Function and regulation of CREB family transcription factors in the nervous system[J].Neuron,2002,35(4):605- 623.

[25] WIGGIN G R,SOLOAGA A,FOSTER J M,et al.MSK1 and MSK2 are required for the mitogen- and stress- induced phosphorylation of CREB and ATF1 in fibroblasts[J].Molecular and Cellular Biology,2002,22(8):2871- 2881.

[26] LIN C H,YEH S H,LIN C H,et al.A role for the PI- 3 kinase signaling pathway in fear conditioning and synaptic plasticity in the amygdala[J].Neuron,2001,31(5):841- 851.

[27] PERKINTON M S,IP J K,WOOD G L,et al.Phosphatidylinositol 3- kinase is a central mediator of NMDA receptor signalling to MAP kinase(Erk1/2),Akt/PKB and CREB in striatal neurones[J].J Neurochem,2002,80(2):239- 254.

[28] STEVENS B,FIELDS R D.Response of Schwann cells to action potentials in development[J].Science,2000,287(5461):2267- 2271.

[29] KIDA S,JOSSELYN S A,PENA DE ORTIZ S,et al.CREB required for the stability of new and reactivated fear memories[J].Nat Neurosci,2002,5(4):348- 355.

[30] ZHOU Y,WON J,KARLSSON M G,et al.CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala[J].Nat Neurosci,2009,12(11):1438- 1443.

[31] BEAUMONT T L,YAO B,SHAH A,et al.Layer- specific CREB target gene induction in human neocortical epilepsy[J].J Neurosci,2012,32(41):14389- 14401.

[32] LOPEZ DE ARMENTIA M,JANCIC D,OLIVARES R,et al.cAMP response element- binding protein- mediated gene expression increases the intrinsic excitability of CA1 pyramidal neurons[J].J Neurosci,2007,27(50):13909- 13918.

[33] LUND I V,HU Y,RAOL Y H,et al.BDNF selectively regulates GABAA receptor transcription by activation of the JAK/STAT pathway[J].Science Signaling,2008,1(41):ra9.

[34] ZHU X,HAN X,BLENDY J A,et al.Decreased CREB levels suppress epilepsy[J].Neurobiol Dis,2012,45(1):253- 263.

[35] ZHU X,DUBEY D,BERMUDEZ C,et al.Suppressing cAMP response element- binding protein transcription shortens the duration of status epilepticus and decreases the number of spontaneous seizures in the pilocarpine model of epilepsy[J].Epilepsia,2015,56(12):1870- 1878.

(本文編輯：周蘭波)

Effect of NMDA receptor on hippocampal astrocyte activation in pilocarpine- induced epileptic mice

YANG Zhan- neng,JIANG Jing- jing,GU Shi- hong,CHEN Lei,LI Hong- wei,ZHU Xin- jian

(MedicalSchoolofSoutheastUniversity,Nanjing210009,China)

Objective: To study the effect of N- methy- D- asparate(NMDA) receptor on hippocampal astrocyte activation in pilocarpine- induced epileptic mice and the possible mechanism involved. Methods: Thirty two C57/BL6 mice were randomly divided into control group, status epilepticu(SE) group, NMDA receptor antagonist MK- 801 treated SE group and MK- 801 alone treated group, with 8 mice in each group. SE epilepsy model was established by intraperitoneal injection of 300 mg·kg-1pilocarpine. Immunohistochemistry was employed to determine hippocampal astrocyte activation in different group of mice. Western Blot was used to detect hippocampal glial fibrillary acidic protein(GFAP) content and transcription factor cAMP response element binding protein(CREB) phosphorylation level. Results: Hippocampal astrocytes were largely activated in SE mice. Immunohistochemistry results showed that GFAP immunostaining intensities in the hippocampus of SE mice were significantly increased as compared to those of control mice. NMDA receptor antagonist MK- 801 treatment after SE, however, suppressed SE- induced hippocampal astrocyte activation. Consistent with the immunohistochemistry results, our Western Blot showed that GFAP level in SE mice was significantly higher than that of control mice. MK- 801 treatment after SE, however, suppressed SE- induced increase of GFAP level. Meanwhile, our Western Blot result showed that phosphorylation level of cAMP response element binding protein(CREB) was significantly increased in SE mice as compared to that of control mice. Blocking NMDA receptor by MK- 801, however, suppressed the SE- induced increase of CREB phosphorylation. Conclusion: Pilocarpine- induced SE can activate hippocampal astrocytes, which are dependent on the activation of NMDA receptors. Meanwhile, hippocampal astrocyte activation in pilocarpine- induced SE mice is accompanied with CREB phosphorylation, suggesting CREB phosphorylation is involved in NMDA receptor- mediated hippocampal astrocytes activation in pilocarpine induced epileptic mice.

N- methy- D- asparate receptors; pilocarpine; epilepsy; hippocampus; astrocyte activation; mice

2016- 09- 24

2016- 12- 05

國家自然科學基金資助項目(81673413)；江蘇省自然科學基金資助項目(BK20141335)；高等學校博士學科點專項科研基金新教師類資助項目(20130092120043)；國家本科生科研訓練計劃(SRTP)項目(201610286134)

楊展能(1994-),男，江蘇淮安人，東南大學醫(yī)學院臨床醫(yī)學專業(yè)在讀學生。E- mail:875211500@qq.com

朱新建 E- mail:xinjianzhu@seu.edu.cn

楊展能,姜靚婧,顧仕紅,等.NMDA受體在匹羅卡品癲癇小鼠海馬星形膠質(zhì)細胞活化中的作用[J].東南大學學報:醫(yī)學版,2017,36(2):129- 136.

R- 33; R742.1

A

1671- 6264(2017)02- 0129- 08

10.3969/j.issn.1671- 6264.2017.02.001