艾厚喜，孫芳玲，侯虹麗,2，張麗，王文

莫諾苷對(duì)大鼠腦缺血再灌注皮層Wnt信號(hào)通路轉(zhuǎn)錄因子表達(dá)的影響①

艾厚喜1，孫芳玲1，侯虹麗1,2，張麗1，王文1

目的研究莫諾苷對(duì)腦缺血再灌注7 d患側(cè)皮層Wnt信號(hào)通路相關(guān)轉(zhuǎn)錄因子神經(jīng)發(fā)生素2(Ngn2)、Pax6、Tbr2表達(dá)的影響。方法健康成年雄性Sprague-Dawley大鼠隨機(jī)分為假手術(shù)組，模型組，莫諾苷小、中、大劑量組，每組3只。線(xiàn)栓法制備大鼠大腦中動(dòng)脈阻塞模型，造模后3 h予莫諾苷30 mg/kg、90 mg/kg、270 mg/kg每天1次灌胃。Western blotting法分析術(shù)后7 d皮層Ngn2、Pax6、Tbr2的表達(dá)。結(jié)果與假手術(shù)組相比，模型組皮層Ngn2表達(dá)升高(P＜0.05)；與模型組相比，莫諾苷中、高劑量組Ngn2表達(dá)明顯升高(P＜0.01)。模型組皮層Pax6的表達(dá)與假手術(shù)組無(wú)顯著性差異，莫諾苷中、高劑量組Pax6表達(dá)升高(P＜0.05)。各組間Tbr2的表達(dá)無(wú)顯著性差異。結(jié)論莫諾苷能增加腦缺血再灌注后皮層Ngn2、Pax6的表達(dá)，提示有促進(jìn)神經(jīng)發(fā)生的作用。

莫諾苷；腦缺血再灌注；轉(zhuǎn)錄因子；Wnt信號(hào)通路；神經(jīng)發(fā)生素2；Pax6；Tbr2；大鼠

[本文著錄格式]艾厚喜，孫芳玲，侯虹麗，等.莫諾苷對(duì)大鼠腦缺血再灌注皮層Wnt信號(hào)通路轉(zhuǎn)錄因子表達(dá)的影響[J].中國(guó)康復(fù)理論與實(shí)踐,2015,21(1):1-4.

CITED AS:Ai HX,Sun FL,Hou HL,et al.Effects of morroniside on Wnt signaling-related transcription factors in ischemic ipsilateral cortex of rats after cerebral ischemia-reperfusion[J].Zhongguo Kangfu Lilun Yu Shijian,2015,21(1):1-4.

腦卒中是全球第三大高致死性、高致殘性疾病[1]。近年來(lái)，對(duì)缺血性腦卒中的病理機(jī)制研究有了很大進(jìn)步，但目前除缺血急性期溶栓治療外，對(duì)于神經(jīng)康復(fù)治療仍沒(méi)有有效的神經(jīng)保護(hù)藥物[2]。研究表明，腦缺血能刺激成年哺乳動(dòng)物內(nèi)源性神經(jīng)發(fā)生[3]，對(duì)腦損傷后期的神經(jīng)修復(fù)有重要意義。但腦損傷所誘發(fā)的神經(jīng)再生相關(guān)因子表達(dá)只在短時(shí)間內(nèi)有明顯變化，自體應(yīng)激產(chǎn)生的神經(jīng)發(fā)生過(guò)程短暫，不足以修復(fù)缺血造成的神經(jīng)血管損傷，恢復(fù)神經(jīng)功能。Wnt/ β-catenin信號(hào)通路是哺乳動(dòng)物體內(nèi)廣泛存在的高度保守的信號(hào)通路，在神經(jīng)系統(tǒng)發(fā)育中發(fā)揮重要作用。研究顯示，Wnt信號(hào)可通過(guò)促進(jìn)祖細(xì)胞增殖，調(diào)節(jié)成年

動(dòng)物側(cè)腦室外側(cè)壁室下區(qū)(subventricular zone,SVZ)內(nèi)的神經(jīng)發(fā)生；在局部腦缺血損傷模型的SVZ區(qū)采用慢病毒表達(dá)Wnt3a-HA能促進(jìn)神經(jīng)功能恢復(fù)，增加紋狀體和SVZ區(qū)成熟神經(jīng)元的數(shù)量，提示W(wǎng)nt信號(hào)可能為腦缺血損傷后干細(xì)胞增殖和分化、神經(jīng)元的存活提供合適的微環(huán)境。神經(jīng)發(fā)生素2(neurogenin 2,Ngn2)、Pax6、Tbr2作為Wnt信號(hào)下游的轉(zhuǎn)錄因子，是神經(jīng)細(xì)胞發(fā)育過(guò)程中所必需的轉(zhuǎn)錄調(diào)控因子(transcriptional control factor,TCF)，參與神經(jīng)元前體細(xì)胞的增殖及分化[4-6]，對(duì)神經(jīng)系統(tǒng)的發(fā)育有著重要的影響。我們的前期研究表明，大鼠大腦中動(dòng)脈阻塞(middle cerebral artery occlusion,MCAO)模型中，Wnt3a和β-catenin蛋白表達(dá)均有顯著變化[7]。

莫諾苷是本室從山茱萸中提取的環(huán)烯醚萜苷類(lèi)單體化合物，已證實(shí)在體外和體內(nèi)具有抗氧化和抗凋亡作用[8-9]。我們通過(guò)缺血性腦卒中大鼠模型的研究，發(fā)現(xiàn)莫諾苷能縮小大腦梗死體積，通過(guò)維持Wnt3a信號(hào)通路的激活，促進(jìn)神經(jīng)干細(xì)胞增殖和分化，最終促進(jìn)神經(jīng)功能恢復(fù)[10]。本研究觀察其對(duì)Wnt信號(hào)通路下游轉(zhuǎn)錄因子表達(dá)的影響，從而探討莫諾苷促神經(jīng)修復(fù)作用的機(jī)制。

1 材料與方法

1.1 主要儀器和試劑

尼龍栓線(xiàn)，直徑0.26 mm(2634-100)：北京沙東生物技術(shù)有限公司。Powerpac Basic電泳儀：美國(guó)Bio-Rad公司。Ngn2抗體、Pax6抗體、Tbr2抗體：美國(guó)ABCAM公司。β-actin抗體：美國(guó)SANTACRUZ公司。

莫諾苷為中藥材山茱萸干燥成熟果肉提取物。高效液相色譜儀分析(C18柱，柱溫35℃，乙腈-水15∶85洗脫糖苷類(lèi)，流速1.0 ml/min，檢測(cè)波長(zhǎng)240 nm)，純度98.5%。

1.2 實(shí)驗(yàn)動(dòng)物分組及給藥

健康成年雄性Sprague-Dawley大鼠，體重260～280 g，由北京維通利華實(shí)驗(yàn)動(dòng)物中心提供，許可證號(hào)：SCXK(京)2006-0009。12 h晝夜循環(huán)清潔級(jí)飼養(yǎng)，預(yù)適應(yīng)環(huán)境1周后進(jìn)行實(shí)驗(yàn)。將大鼠編號(hào)，用隨機(jī)數(shù)字表法分為假手術(shù)組，模型組，莫諾苷小、中、大劑量組，每組至少3只。

莫諾苷溶于生理鹽水，在MCAO造模后3 h按照30 mg/kg、90 mg/kg、270 mg/kg每天1次灌胃給藥，連續(xù)7 d。假手術(shù)組和模型組予等體積蒸餾水。

1.3 模型制備

模型制作參考Longa線(xiàn)栓法[11]。大鼠造模前一晚禁食，自由飲水。10%水合氯醛350 mg/kg腹腔注射麻醉。仰臥位固定，頸部消毒，切開(kāi)右側(cè)頸部，逐層分離暴露右側(cè)頸總動(dòng)脈、頸外動(dòng)脈以及頸內(nèi)動(dòng)脈，分別結(jié)扎右側(cè)頸總動(dòng)脈及右側(cè)頸外動(dòng)脈。在頸總動(dòng)脈近動(dòng)脈分叉處剪一小口，將線(xiàn)栓插入左側(cè)頸內(nèi)動(dòng)脈約18～20 mm，到達(dá)狹窄的近端大腦前動(dòng)脈，感到輕微阻力時(shí)立即停止插線(xiàn)?？p合傷口，抗生素預(yù)防感染，留置線(xiàn)頭于體外。30 min后，拔出尼龍線(xiàn)，完成再灌注。假手術(shù)組麻醉后進(jìn)行相同的手術(shù)操作，但不插線(xiàn)。

術(shù)后控制室溫25～30℃，3 h動(dòng)物清醒后觀察其行為。采用Ludmila Belayev評(píng)分法[12]。①姿勢(shì)反射試驗(yàn)，即提尾懸空試驗(yàn)：無(wú)明顯神經(jīng)功能缺失為0分，梗死對(duì)側(cè)肢體屈曲為1分，側(cè)推試驗(yàn)陽(yáng)性為2分。②前肢放置試驗(yàn)。A.視覺(jué)亞試驗(yàn)。前方刺激：實(shí)驗(yàn)者將動(dòng)物握于手中，使其前爪懸空，讓動(dòng)物位于桌子前方，自桌面上方10 cm處向桌面緩慢斜線(xiàn)靠近，大鼠正常反應(yīng)為前肢抓向桌面，損傷大鼠則表現(xiàn)為肢體反應(yīng)延遲。0分，動(dòng)物肢體放置反應(yīng)正常；1分，反應(yīng)延遲不超過(guò)2 s；2分，反應(yīng)延遲超過(guò)2 s。側(cè)方刺激：讓動(dòng)物位于桌子側(cè)方，實(shí)驗(yàn)檢測(cè)方法及評(píng)分標(biāo)準(zhǔn)同前方刺激。B.觸覺(jué)亞實(shí)驗(yàn)。將動(dòng)物雙眼遮住，并使其前爪懸空，用其前爪背刺輕觸桌面，刺激深度僅達(dá)皮膚和毛發(fā)，動(dòng)物反應(yīng)及評(píng)分同視覺(jué)試驗(yàn)，同樣分為前方和側(cè)方刺激。C.本體覺(jué)亞實(shí)驗(yàn)。操作及評(píng)分同觸覺(jué)亞實(shí)驗(yàn)，予前爪較大壓力，使刺激深達(dá)肌肉及關(guān)節(jié)。

1.4 Western blotting

10%水合氯醛0.4 ml/kg腹腔注射麻醉，開(kāi)顱取腦，取皮層梗死周邊區(qū)組織，加入7倍腦皮層質(zhì)量預(yù)冷RIPA裂解緩沖液勻漿。勻漿液14000 g 4℃離心30 min，收集上清。BCA法測(cè)定蛋白濃度，用裂解液調(diào)節(jié)使得各組總蛋白濃度一致，加緩沖液變性煮沸10 min。

待檢測(cè)樣品通過(guò)10%～15%十二烷基磺酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)。轉(zhuǎn)膜后將NC膜浸在5%脫脂奶粉，搖床室溫封閉2 h，分別加入Ngn2、Pax6、Tbr2一抗4℃過(guò)夜，洗掉多余一抗后加入辣根過(guò)氧化酶標(biāo)記的二抗室溫孵育2 h，洗掉多余二抗。ECL化學(xué)發(fā)光法顯色，曝光。用Quantity One軟件對(duì)顯影條帶進(jìn)行灰度分析，相同泳道中蛋白含量用β-ac-

tin水平標(biāo)化。

1.5 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

腦缺血再灌注7 d后，模型組患側(cè)皮層Ngn2表達(dá)較假手術(shù)組升高(P＜0.05)，但Pax6和Tbr2表達(dá)與假手術(shù)組無(wú)顯著性差異。莫諾苷中、大劑量組患側(cè)皮層Ngn2和Pax6表達(dá)較模型組升高(均P＜0.05)。各組患側(cè)皮層Tbr2表達(dá)無(wú)顯著性差異。見(jiàn)表1。

表1 各組患側(cè)皮層Ngn2、Pax6、Tbr2光密度比較(/β-actin)

3 討論

Wnt/β-catenin信號(hào)可介導(dǎo)細(xì)胞存活、神經(jīng)干細(xì)胞的增殖及分化等活動(dòng)[13-14]，參與成年神經(jīng)發(fā)生過(guò)程，在腦卒中等疾病的神經(jīng)修復(fù)中起重要作用。研究顯示，腦卒中后該通路各元件在大腦SVZ區(qū)的表達(dá)上調(diào)，并且通過(guò)激活下游靶基因，在促進(jìn)缺血損傷后的神經(jīng)發(fā)生中發(fā)揮重要作用[14-17]。

轉(zhuǎn)錄因子Ngn2、Pax6、Tbr2已被證實(shí)為Wnt通路介導(dǎo)的成年神經(jīng)發(fā)生的下游介質(zhì)[17-18]。轉(zhuǎn)錄因子Pax6在大部分增殖的SVZ區(qū)前體細(xì)胞表達(dá)，并在遷移的成神經(jīng)細(xì)胞亞群中促進(jìn)神經(jīng)元的分化[18]。對(duì)Pax6突變鼠的研究顯示，Pax6通過(guò)Wnt-Tcf信號(hào)通路調(diào)節(jié)前腦發(fā)育[19]，并且在嗅球部位調(diào)節(jié)內(nèi)在神經(jīng)元特殊亞型的分化[20]。Tbr2是含有T-box結(jié)構(gòu)的轉(zhuǎn)錄因子，對(duì)成年小鼠SVZ區(qū)和海馬齒狀回的神經(jīng)發(fā)生非常重要[21]。Tbr2在SVZ區(qū)中間神經(jīng)元前體細(xì)胞和基底祖細(xì)胞表達(dá)，為皮層上部產(chǎn)生神經(jīng)元[22]。Tbr2功能性失活可促進(jìn)神經(jīng)干細(xì)胞增殖，但長(zhǎng)時(shí)間Tbr2缺失，則由于神經(jīng)元分化而導(dǎo)致神經(jīng)發(fā)生停止[19]。Ngn2是具有基本螺旋-環(huán)-螺旋結(jié)構(gòu)(bHLH)轉(zhuǎn)錄因子的一員，可抑制星形膠質(zhì)細(xì)胞分化，促進(jìn)神經(jīng)元分化。Berninger等通過(guò)逆轉(zhuǎn)錄病毒將Ngn2基因?qū)氪笫竽X皮質(zhì)的星形膠質(zhì)細(xì)胞，表明Ngn2可促進(jìn)神經(jīng)蛋白表達(dá)，使膠質(zhì)細(xì)胞分化為神經(jīng)元[23]。已有研究顯示，腦缺血可激活Ngn2基因表達(dá)[24]，而且β-catenin信號(hào)能上調(diào)Ngn2表達(dá)，但具體機(jī)制還不清楚[25-26]。

本研究顯示，大鼠腦缺血再灌注后，患側(cè)皮層轉(zhuǎn)錄因子Ngn2表達(dá)增加；給予莫諾苷治療可使Ngn2和Pax6表達(dá)進(jìn)一步上調(diào)，提示莫諾苷可能通過(guò)調(diào)控該因子表達(dá)，促進(jìn)神經(jīng)干細(xì)胞的增殖，并進(jìn)一步介導(dǎo)神經(jīng)元分化。

缺血再灌注大鼠患側(cè)皮層Tbr2表達(dá)無(wú)明顯變化。我們推測(cè)可能與Pax6和Tbr2表達(dá)模式的復(fù)雜性有關(guān)[27-28]。有研究表明，Pax6和Tbr2的作用相互關(guān)聯(lián)。發(fā)育過(guò)程中Pax6的高表達(dá)可導(dǎo)致有絲分裂后期細(xì)胞分化為中間神經(jīng)元，但下調(diào)Pax6又可促進(jìn)新皮層嗅球內(nèi)僧帽細(xì)胞Tbr2的表達(dá)[28]。我們將進(jìn)一步通過(guò)分子生物學(xué)方法明確莫諾苷通過(guò)Wnt/β-catenin信號(hào)通路促進(jìn)神經(jīng)發(fā)生的具體機(jī)制，為將莫諾苷開(kāi)發(fā)成為腦卒中的神經(jīng)保護(hù)藥物提供實(shí)驗(yàn)依據(jù)。

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Effects of Morroniside on Wnt Signaling-related Transcription Factors in Ischemic Ipsilateral Cortex of Rats after Cerebral Ischemia-reperfusion

AI Hou-xi,SUN Fang-ling,HOU Hong-li,ZHANG Li,WANG Wen.Xuanwu Hospital of Capital Medical University, Key Laboratory for Neurodegenerative Diseases of Ministry of Education,Beijing 100053,China

Objective To study the effects of morroniside on the expression of Wnt signaling-related transcription factors neurogenin 2 (Ngn2),Pax6 and Tbr2 in the ischemic ipsilateral cortex 7 days after cerebral ischemia-reperfusion in rats.Methods 15 male Sprague-Dawley rats were randomly divided into sham group(n=3),ischemia group(n=3),and morroniside groups(low,medium and high dosage groups,n=3).The middle cerebral artery were occluded for 30 min,and re-perfused.Morroniside was administered intragastrically once a day at dose of 30 mg/kg,90 mg/kg and 270 mg/kg 3 hours after operation.The expression of Ngn2,Pax6 and Tbr2 in the ischemic ipsilateral cortex were detected with Western blotting analysis 7 days after operation.Results The expression of Ngn2 increased in the ischemia group compared with the sham group(P＜0.05),and it further increased the morroniside groups of medium and high dosage compared with the ischemia group(P＜0.01).There was no significant difference between the ischemia group and sham group in the expression of Pax6, while it increased the morroniside groups of medium and high dosage compared with the ischemia group(P＜0.01).There was no significant difference among all the groups in the expression of Tbr2.Conclusion Morroniside could increase the expression of Ngn2 and Pax6 in the ischemic ipsilateral cortex 7 days after ischemia-reperfusion in rats,suggesting promoting the neurogenesis after ischemia.

morroniside;cerebral ischemia-reperfusion;transcription factors;Wnt signaling;neurogenin 2;Pax 6;Tbr2;rats

10.3969/j.issn.1006-9771.2015.01.001

R743.3

A

1006-9771(2015)01-0001-04

2014-11-15

2014-12-01)

1.“重大新藥創(chuàng)制”科技重大專(zhuān)項(xiàng)(No.2012ZX09102201-016)；2.國(guó)家自然科學(xué)基金項(xiàng)目(No.81173575；No.81373994)；3.首都醫(yī)科大學(xué)宣武醫(yī)院院級(jí)課題。

1.首都醫(yī)科大學(xué)宣武醫(yī)院動(dòng)物實(shí)驗(yàn)室，北京市老年病醫(yī)療研究中心，北京腦重大疾病研究院，北京市100053；2.河北北方學(xué)院，河北張家口市075000。作者簡(jiǎn)介：艾厚喜(1956-)，男，滿(mǎn)族，北京市人，主管技師，主要研究方向：神經(jīng)藥理，中藥藥理。通訊作者：王文。E-mail:lzwwang@163.com。