千超，劉鋒，肖學(xué)謙，李峰，高喜松，黨連鋒，張毓

有絲分裂原活化蛋白激酶信號通路介導(dǎo)大鼠星形膠質(zhì)細(xì)胞氧糖剝奪后水通道蛋白4的表達(dá)①

千超，劉鋒，肖學(xué)謙，李峰，高喜松，黨連鋒，張毓

目的探討有絲分裂原活化蛋白激酶(MAPKs)是否介導(dǎo)缺血后大鼠星形膠質(zhì)細(xì)胞水通道蛋白4(AQP4)的表達(dá)。方法分離培養(yǎng)新生Sprague-Dawley大鼠腦星形膠質(zhì)細(xì)胞。第2代細(xì)胞分成對照組、氧糖剝奪(OGD)組和阻斷組。后兩組復(fù)制OGD 5 h后復(fù)氧模型，12 h后阻斷組分別換入含U0126(1 μmol/L和10 μmol/L,U1組和U10組)、SP600125(1 μmol/L和10 μmol/L,SP1組和SP10組)和SB203580(1 μmol/L和10 μmol/L,SB1組和SB10組)的培養(yǎng)液。復(fù)氧后0.5 h、1 h、1.5 h、2 h、3 h、4 h、8 h和12 h檢測OGD組細(xì)胞體積，復(fù)氧后0.5 h、1 h、1.5 h、8 h和12 h Western blotting法檢測OGD組AQP4表達(dá)；復(fù)氧后24 h，檢測各組乳酸脫氫酶(LDH)活性，Western blotting法檢測各組AQP4，磷酸化細(xì)胞外調(diào)節(jié)蛋白激酶(p-ERK)、c-Jun氨基末端激酶(p-JNK)和p38 MAPK(p-p38 MAPK)表達(dá)。結(jié)果復(fù)氧后1.5 h、2 h、3 h和4 h時，OGD組細(xì)胞體積較對照組顯著增大(P＜0.001)，OGD組AQP4水平較對照組顯著升高(P＜0.001)，并在復(fù)氧后1.5 h達(dá)到峰值。OGD組p-ERK、p-JNK和p-p38 MAPK、AQP4水平較對照組顯著增加(P＜0.001)，阻斷組p-ERK、p-JNK和p-p38 MAPK較OGD組顯著下降(P＜0.001)，SB10組AQP4較OGD組顯著下降(P＜0.001)。除SP1組、SB1組外，各干預(yù)組LDH活性較OGD組顯著下降(P＜0.01)，SB10組最低(P＜0.001)。結(jié)論MAPK信號通路，特別是p38 MAPK可介導(dǎo)大鼠星形膠質(zhì)細(xì)胞AQP4蛋白表達(dá)，加重細(xì)胞壞死。

氧糖剝奪；星形膠質(zhì)細(xì)胞；有絲分裂原活化蛋白激酶；信號通路；水通道蛋白4；水腫；大鼠

水通道蛋白4(aquaporins 4,AQP4)在中樞神經(jīng)系統(tǒng)的水運輸中起重要作用[1-2]，并在腦星形膠質(zhì)細(xì)胞中高表達(dá)[3]。近年研究表明，調(diào)節(jié)星形膠質(zhì)細(xì)胞AQP4的表達(dá)可能是治療腦水腫的新策略[4]。絲裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)是調(diào)節(jié)細(xì)胞滲透壓的重要信號通路，動物研究表明，缺血性腦組織中MAPKs的表達(dá)及其磷酸化水平發(fā)生變化[5-6]；高滲應(yīng)激可通過p38 MAPK途徑增加大鼠星形膠質(zhì)細(xì)胞AQP4的表達(dá)[7]。但也有學(xué)者得出相反結(jié)果[8]。本研究觀察MAPK信號通路與腦水腫后星形膠質(zhì)細(xì)胞AQP4調(diào)節(jié)的關(guān)系。

1 材料與方法

1.1 材料

新生Sprague-Dawley大鼠5只(SPF級)：西安交通大學(xué)醫(yī)學(xué)實驗動物中心。無酚紅細(xì)胞培養(yǎng)液、胎牛血清、U0126和SP600125：SIGMA公司。PBS緩沖液和胰蛋白酶：GIBCO公司。SB203580：BIOMOL公司。膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein,GFAP)、AQP4、磷酸化 p38 MAPK(phosphorylation of p38 MAPK,p-p38 MAPK)、磷酸化細(xì)胞外調(diào)節(jié)蛋白激酶(phosphorylation of extracellular regulated protein kinases,p-ERK)、磷酸化c-Jun氨基末端激酶(phosphorylation of c-Jun N-terminal kinase,p-JNK)和β-actin抗體：CELL SIGNAL TECHNOLOGY公司。乳酸脫氫酶(lactic dehydrogenase,LDH)活性檢測試劑盒：廣州碧云天公司。蛋白定量試劑盒：PIERCE公司。激光共聚焦顯微鏡：徠卡公司。酶標(biāo)儀和電泳儀：BIO-RAD公司。生物安全柜和細(xì)胞培養(yǎng)箱：THERMO公司。

1.2 星形膠質(zhì)細(xì)胞的培養(yǎng)與鑒定

無菌條件下分離大鼠大腦皮質(zhì)，0.25%胰蛋白酶37℃水浴消化15 min，離心，收集細(xì)胞，接種于含10%胎牛血清的DMEM-F12培養(yǎng)基，5%CO2、37℃培養(yǎng)箱培養(yǎng)[7,9]。

取第2代細(xì)胞接種于細(xì)胞培養(yǎng)板，甲醛固定，PBS緩沖液漂洗3次，加入GFAP抗體100 μl，4℃孵育過夜。PBS漂洗3次，加FITC熒光標(biāo)記二抗150 μl，37℃孵育1 h；PBS洗滌3次，封片，熒光顯微鏡下觀察。

1.3 造模方法

取第2代星形膠質(zhì)細(xì)胞，以每孔5×105濃度接種于6孔細(xì)胞培養(yǎng)板，并做細(xì)胞爬片。將細(xì)胞分成對照組、OGD組和阻斷組。后兩組參考文獻(xiàn)復(fù)制氧糖剝奪(oxygen-glucose deprivation,OGD)和復(fù)氧模型[10]。第2代星形膠質(zhì)細(xì)胞培養(yǎng)24 h后，棄去培養(yǎng)液，PBS緩沖液漂洗2次，加入不含葡萄糖的無血清DMEM培養(yǎng)液，95%N2、5%CO2、37℃培養(yǎng)5 h。然后換入正常細(xì)胞培養(yǎng)液，5%CO2、37℃培養(yǎng)。12 h后，阻斷組分別換入含 U0126(1 μmol/L 和 10 μmol/L,U1 組和U10組)、SP600125(1 μmol/L和10 μmol/L,SP1組和SP10組)和SB203580(1 μmol/L和10 μmol/L,SB1組和SB10組)的培養(yǎng)液。其他兩組正常換液。

1.4 細(xì)胞體積檢測

復(fù)氧后0.5 h、1 h、1.5 h、2 h、3 h、4 h、8 h和12 h，細(xì)胞培養(yǎng)液加1 mmol/L甲基葡萄糖(3-O-methylglucose,3-OMG)和0.5 μCi/ml的3H-3-OMG，繼續(xù)培養(yǎng)12 h后收集上清，進(jìn)行放射性檢測[7]。細(xì)胞用4℃預(yù)冷緩沖液(pH 7.4)漂洗后，用1 N NaOH 0.5 ml裂解，按照試劑盒說明書進(jìn)行檢測蛋白含量。

1.5 LDH活性檢測

復(fù)氧后24 h，取各組細(xì)胞培養(yǎng)液，4℃預(yù)冷PBS緩沖液漂洗3遍，加入LDH工作液60μl混勻，室溫(約25℃)避光孵育30 min，450 nm處測定吸光度。

1.6 免疫熒光染色

加入阻斷劑后，各組繼續(xù)培養(yǎng)2 h，棄培養(yǎng)液，4℃預(yù)冷PBS洗3次，每孔加入4%多聚甲醛固定20 min，PBS洗3次。0.3%Triton X-100透化。加AQP4一抗(1∶400)4℃孵育過夜，PBS洗3次，加Alexa標(biāo)記二抗和TRITC標(biāo)記鬼筆環(huán)肽，37℃孵育1 h，PBS緩沖液清洗3次，封片，激光共聚焦顯微鏡下觀察。

1.7 Western blotting

各組棄去培養(yǎng)液，PBS漂洗3次，加入RIPA細(xì)胞裂解液800μl裂解30 min。12,000 r/min離心15 min，收集上清，95℃變性5 min。各組取蛋白質(zhì)樣品10 μg，15%SDS-PAGE電泳分離，轉(zhuǎn)移至PVDF膜，2%脫脂奶粉封閉2 h，加入AQP4、p-p38 MAPK、p-ERK、p-JNK和β-actin一抗，4℃過夜。PBST搖床漂洗3次，每次10 min，加入二抗，37℃孵育2 h。增強化學(xué)發(fā)光法檢測目的蛋白，用Image-Pro plus6.0軟件測定灰度值。

復(fù)氧后0.5 h、1 h、1.5 h、8 h和12 h檢測OGD組AQP4表達(dá)；復(fù)氧后24 h檢測各組AQP4、p-p38 MAPK、p-ERK、p-JNK表達(dá)。

1.8 統(tǒng)計學(xué)分析

采用SPSS 20.0進(jìn)行數(shù)據(jù)處理。檢測結(jié)果以(xˉ±s)表示，多組均數(shù)比較采用單因素方差分析(one-way ANOVA)，組間比較選用LSDt檢驗法。顯著性水平α1=0.05，非常顯著性水平α2=0.01。

2 結(jié)果

2.1 星形膠質(zhì)細(xì)胞鑒定

細(xì)胞GFAP陽性，胞體較大，具有短而粗大的突起，形態(tài)不規(guī)則，輪廓較清晰，為星形膠質(zhì)細(xì)胞。OGD后，細(xì)胞體積增大。見圖1。

2.2 OGD復(fù)氧后星形膠質(zhì)細(xì)胞改變

復(fù)氧1.5 h、2 h、3 h和4 h時，細(xì)胞體積和對照組相比顯著增大(P＜0.001)，尤以1.5 h時細(xì)胞體積最大。見表1。

復(fù)氧后，星形膠質(zhì)細(xì)胞AQP4表達(dá)較對照組顯著升高(P＜0.001)，復(fù)氧后1.5 h達(dá)到峰值。見圖2、表2。

2.3 LDH活性

OGD組LDH活性顯著高于對照組(P＜0.001)；各阻斷組除SB1組外，LDH活性均較OGD組下降(P＜0.05)，SB10組最低(P＜0.001)。見表3。

2.4 Western blotting

復(fù)氧后，OGD組p-ERK、p-JNK、p-p38 MAPK和AQP4水平均較對照組顯著升高，阻斷組p-ERK、p-JNK和p-p38 MAPK均較OGD組顯著下降(P＜0.001)，SB10 組 AQP4 水平最低(P＜0.001)。見圖 3、圖4、表4、表5。

2.5 免疫熒光染色

有較多AQP4蛋白熒光信號，SB10組較OGD組明顯減少(圖5)。

表1 復(fù)氧后各時間點細(xì)胞體積(μl/mg)

圖1 星形膠質(zhì)細(xì)胞培養(yǎng)

圖2 復(fù)氧后各時間點AQP4蛋白表達(dá)(Western blotting)

圖3 各組p-ERK、p-JNK、p-p38 MAPK蛋白表達(dá)(Western blotting)

圖4 各組AQP4蛋白表達(dá)(Western blotting)

表2 復(fù)氧后各時間點AQP4蛋白表達(dá)(灰度)

表3 復(fù)氧后各組LDH活性(%)

表4 各組p-ERK、p-JNK、p-p38 MAPK蛋白表達(dá)(灰度)

表5 各組AQP4蛋白表達(dá)(灰度)

3 討論

本研究顯示，星形膠質(zhì)細(xì)胞OGD水腫后，AQP4表達(dá)增加；阻斷MAPK通道各個因子，均可抑制AQP4異常高表達(dá)，尤以p38 MAPK特異性抑制劑SB203580效果最佳。本研究還顯示，SB203580可以減少水腫后星形膠質(zhì)細(xì)胞死亡，與最新報道一致[11]；可以減少星形膠質(zhì)細(xì)胞水腫，與Nito等[12]的研究結(jié)果一致。提示星形膠質(zhì)細(xì)胞OGD后AQP4蛋白的表達(dá)主要與p38 MAPK信號通路有關(guān)，Qi等[13]也得到相似的結(jié)論。

圖5 星形膠質(zhì)細(xì)胞AQP4蛋白表達(dá)(免疫熒光染色，100×)

JNK特異性抑制劑SP600125和ERK特異性抑制劑U0126都可以減少AQP4蛋白的表達(dá)，但對細(xì)胞凋亡的作用較小。我們分析可能是SP600125只能阻斷JNK1表達(dá)，而對JNK2/3無效。有學(xué)者發(fā)現(xiàn)SP600125可以抑制腦缺血后神經(jīng)元的凋亡[8]，但可能對本研究涉及的星形膠質(zhì)細(xì)胞作用不大。

大量資料表明，腦卒中、腦外傷、腦膜炎和腦腫瘤患者，腦組織中星形膠質(zhì)細(xì)胞AQP4均高表達(dá)[14-19]。Frydenlund等[20]發(fā)現(xiàn)，腦動脈閉塞再灌注24 h時，AQP4在梗死周圍皮質(zhì)高表達(dá)。體內(nèi)實驗也表明，p38 MAPK信號通路在腦卒中后被激活，且短暫性腦缺血后，半暗帶區(qū)域星形膠質(zhì)細(xì)胞誘導(dǎo)p38 MAPK的延遲激增。Piao等[21]發(fā)現(xiàn)，短暫性腦缺血后，經(jīng)SB203580干預(yù)，可以減少梗死面積和神經(jīng)元死亡，有效保護(hù)腦組織。

Tait等[22]卻認(rèn)為，AQP4缺失可能通過減少腦部過量水分的消除，增加蛛網(wǎng)膜下腔出血后腦水腫。Manley等[23]也證明，AQP4敲除小鼠腦缺血后水腫減少。這些差異可能是由于使用的模型不同所致。需要對AQP4敲除小鼠腦損傷后的水轉(zhuǎn)運進(jìn)行更詳細(xì)的研究和分析。

腦損傷后AQP4的異常表達(dá)不僅與p38 MAPK信號通路有關(guān)，也與蛋白激酶C和核因子κB信號通路相關(guān)。Arima等[24]研究表明，p38 MAPK信號通路是誘發(fā)AQP4異常表達(dá)的必然通路，但不是唯一通路。本研究發(fā)現(xiàn)，大鼠腦細(xì)胞損傷后AQP4的異常表達(dá)與p38 MAPK、ERK和JNK都有關(guān)，但與p38 MAPK關(guān)系最密切。p38 MAPK信號通路阻斷劑SB203580能夠抑制星形膠質(zhì)細(xì)胞水腫后細(xì)胞死亡。這為臨床治療神經(jīng)損傷水腫提供了基礎(chǔ)。有待進(jìn)一步研究。

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Role of Mitogen-activated Protein Kinase Pathways in Expression of Aquaporin-4 in Astrocytes after Oxygen-glucose Deprivation in Rats

QIAN Chao,LIU Feng,XIAO Xue-qian,LI Feng,GAO Xi-song,DANG Lian-feng,ZHANG Yu
Deparment of Neurosurgery,No.215 Hospital of Shaanxi Nuclear Industry,Xianyang,Shaanxi 712000,China
Correspondence toLIU Feng.E-mail:liufeng2112010@163.com

ObjectiveTo investigate whether mitogen-activated protein kinases(MAPKs),which were involved in changes in osmolality,might mediate aquaporin-4(AQP4)expression in astrocytes after oxygen-glucose deprivation(OGD)in rats.MethodsAstrocytes were obtained from new born Sprague-Dawley rats.The P2cells were divided into control group,OGD group and inhibitors of U0126,SB203580 and SP600125 groups.The latter groups underwent OGD for five hours and reoxygenated,the inhibitors of U0126,SB203580 and SP600125(1 μmol/L and 10 μmol/L,respectively)groups,named U1,U10,SB1,SB10,SP1 and SP10 groups,respectively,were cultured with the inhibitors for twelve hours.The volume of cells in OGD group was measured half,one,one and half,two,three,four,eight and twelve hours after reoxygenation,and the expression of AQP4 was detected half,one,one and half,eight and twelve hours after reoxygenation with Western blotting.The expression ofAQP4,and phosphorylation of extracellular regulated protein kinases(p-ERK),c-Jun N-terminal kinase(p-JNK)and p38 MAPK(p-p38 MAPK)was detected 24 hours after reoxygenation in all the groups,while the activity of lactate dehydrogenase(LDH)was measured.ResultsThe volume of cells increased in OGD group one and half,two,three and four hours after reoxygenation compared with those in the control group(P＜0.001),and the expression of AQP4 also increased in OGD group after reoxygenation(P＜0.001),especially 1.5 hours of reoxygenation.The expression of AQP4,p-ERK,p-JNK and p-p38 MAPK increased in OGD group after five hours of OGD compared with those in the control group(P＜0.001),and the expression of p-ERK,p-JNK and p-p38 MAPK decreased in the inhibitors groups compared with those in OGD group(P＜0.001),and the expression of AQP4 decreased in SB10 group(P＜0.001).The activity of LDH was less in all the inhibitors groups except SP1 and SB1 groups than in OGD group(P＜0.01),and was the least in SB10 group(P＜0.001).ConclusionMAPKs signal pathway,especially p38 MAPK,may promote AQP4 expression in astrocytes after OGD in rat,and play a role in cells death.

陜西省核工業(yè)215醫(yī)院神經(jīng)外科，陜西咸陽市712000。作者簡介：千超(1974-)，男，漢族，陜西咸陽市人，碩士，副主任醫(yī)師，主要研究方向：顱底腫瘤的分子機制。通訊作者：劉鋒。E-mail:liufeng2112010@163.com。

10.3969/j.issn.1006-9771.2017.12.006

oxygen-glucose deprivation;astrocyte;mitogen-activated protein kinases;signal pathway;aquaporin-4;edema;rats

R742

A

1006-9771(2017)12-1397-06

[本文著錄格式]千超，劉鋒，肖學(xué)謙，等.有絲分裂原活化蛋白激酶信號通路介導(dǎo)大鼠星形膠質(zhì)細(xì)胞氧糖剝奪后水通道蛋白4的表達(dá)[J].中國康復(fù)理論與實踐,2017,23(12):1397-1402.

CITED AS:Qian C,Liu F,Xiao XQ,et al.Role of mitogen-activated protein kinase pathways in expression of aquaporin-4 in astrocytes after oxygen-glucose deprivation in rats[J].Zhongguo Kangfu Lilun Yu Shijian,2017,23(12):1397-1402.

2017-04-02

2017-06-08)