尹曉敏 徐 婷 戴伍飛 錢嘉逸 陳 晨 張 蘭 李 林*

(1.首都醫(yī)科大學(xué)宣武醫(yī)院藥物研究室 北京市神經(jīng)藥物工程技術(shù)研究中心，北京 100053；2.南通大學(xué)醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)系，江蘇南通 226001)

·神經(jīng)系統(tǒng)疾病的基礎(chǔ)和臨床研究·

二苯乙烯苷對(duì)神經(jīng)細(xì)胞淀粉樣肽前體蛋白表達(dá)的影響及其與CREB調(diào)節(jié)相關(guān)的作用機(jī)制

尹曉敏1,2徐 婷2戴伍飛2錢嘉逸2陳 晨1張 蘭1李 林1*

(1.首都醫(yī)科大學(xué)宣武醫(yī)院藥物研究室 北京市神經(jīng)藥物工程技術(shù)研究中心，北京 100053；2.南通大學(xué)醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)系，江蘇南通 226001)

目的探討二苯乙烯苷(tetrahydroxystilbene glucoside,TSG)對(duì)培養(yǎng)神經(jīng)細(xì)胞淀粉樣肽前體蛋白(amyloid precursor protein，APP)表達(dá)的影響及其作用機(jī)制。方法應(yīng)用Western blotting法檢測(cè)APP和cAMP效應(yīng)元件結(jié)合蛋白(cAMP responsive element-binding protein,CREB)的表達(dá)。用Matinspector軟件分析APP啟動(dòng)子區(qū)CRE順式反應(yīng)元件；構(gòu)建不同長(zhǎng)度APP啟動(dòng)子區(qū)熒光素酶表達(dá)質(zhì)粒；用雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)細(xì)胞中熒光素酶活性。結(jié)果由Forskolin激活SH-SY5Y細(xì)胞中CREB的同時(shí)可下調(diào)APP表達(dá)，成功構(gòu)建了含有CRE元件的APP啟動(dòng)子區(qū)熒光素酶表達(dá)質(zhì)粒。TSG能夠抑制SH-SY5Y細(xì)胞內(nèi)APP表達(dá)，并對(duì)APP啟動(dòng)子轉(zhuǎn)染細(xì)胞內(nèi)含有CRE1和2的APP熒光素酶報(bào)告基因質(zhì)粒表達(dá)有顯著抑制作用。結(jié)論TSG能夠下調(diào)神經(jīng)細(xì)胞中APP的表達(dá)，其機(jī)制可能與影響CREB與CRE元件的結(jié)合有關(guān)。

二苯乙烯苷；淀粉樣肽前體蛋白；cAMP效應(yīng)元件結(jié)合蛋白；阿爾茨海默病

阿爾茨海默病(Alzheimer’s disease，AD)是老年人中最常見的神經(jīng)退行性疾病，其患者腦內(nèi)主要病理表現(xiàn)為細(xì)胞內(nèi)大量的神經(jīng)原纖維纏結(jié)的形成以及細(xì)胞外大量β-淀粉樣肽(β-amyloid,Aβ)聚集形成的老年斑[1]。其中，Aβ是由淀粉樣肽前體蛋白(amyloid precursor protein,APP)經(jīng)成淀粉樣肽途徑生成的小分子毒性片段[2]。有文獻(xiàn)[3]顯示，APP表達(dá)水平與可溶性Aβ水平增高呈正相關(guān)。

cAMP效應(yīng)元件結(jié)合蛋白(cAMP responsive element-binding protein,CREB)在真核生物正常的生理調(diào)節(jié)下可調(diào)控其靶基因的轉(zhuǎn)錄。CREB可以特異性地結(jié)合到cAMP效應(yīng)元件(cAMP responsive element，CRE)上。CRE廣泛存在于真核生物基因啟動(dòng)子區(qū)，CREB通過與CRE結(jié)合，可以影響下游靶基因的轉(zhuǎn)錄效率。CREB在神經(jīng)元生成、突觸可塑性以及學(xué)習(xí)記憶等方面都具有非常重要的調(diào)節(jié)作用[4-6]，可作為神經(jīng)系統(tǒng)疾病的潛在藥物靶點(diǎn)。本研究中筆者證實(shí)了CREB對(duì)APP表達(dá)的調(diào)節(jié)作用。

二苯乙烯苷(2,3,5,4′-tetrahydroxystilbene-2-O-b-D-glucoside,TSG)是中藥何首烏的主要有效成分和標(biāo)志成分。本課題組前期在APP轉(zhuǎn)基因小鼠模型中，發(fā)現(xiàn)TSG能夠改善模型鼠的學(xué)習(xí)記憶能力，減少腦顳葉皮質(zhì)淀粉樣斑塊的數(shù)目和面積，減低皮質(zhì)和海馬Aβ的量[7-9]。但是TSG對(duì)APP表達(dá)的影響及其作用機(jī)制尚未見報(bào)道。本文通過構(gòu)建APP啟動(dòng)子區(qū)雙熒光素酶報(bào)告基因質(zhì)粒，研究TSG對(duì)神經(jīng)細(xì)胞內(nèi)APP蛋白表達(dá)的影響，并探討其與CREB調(diào)節(jié)相關(guān)的分子機(jī)制。

1 材料與方法

1.1 藥物與試劑

TSG購自中國食物與藥物研究所，純度>98%；Forskolin購自美國Sigma-aldrich公司，純度>98%?？笰PP抗體購自德國Merck-Millipore公司，CREB及磷酸化CREB抗體購自美國Cell signaling公司，GAPDH抗體購自美國Santa cruz公司。蛋白標(biāo)準(zhǔn)品購自美國Bio-rad公司；DNA標(biāo)準(zhǔn)品購自北京全式金生物技術(shù)有限公司。

1.2 細(xì)胞培養(yǎng)

人神經(jīng)母細(xì)胞瘤細(xì)胞系SH-SY5Y細(xì)胞培養(yǎng)在含10%(體積分?jǐn)?shù))胎牛血清的DMEM/F12培養(yǎng)基中，置37 ℃含5%(體積分?jǐn)?shù))CO2的培養(yǎng)箱內(nèi)。人胚胎腎細(xì)胞系HEK-293FT細(xì)胞培養(yǎng)在含10%(體積分?jǐn)?shù))胎牛血清的高糖DMEM培養(yǎng)基中，置37 ℃含5%(體積分?jǐn)?shù))CO2的培養(yǎng)箱，每?jī)商鞊Q一次培養(yǎng)基。

1.3 Western blotting分析

細(xì)胞裂解后，細(xì)胞總蛋白經(jīng)聚丙烯酰胺凝膠電泳(SDS-PAGE)分離，然后電轉(zhuǎn)至聚偏氟乙烯(PVDF)膜。用5%(質(zhì)量分?jǐn)?shù))脫脂牛奶封閉30 min，一抗(抗APP、CREB或p-CREB抗體)在室溫下孵育過夜。然后用含0.5% (體積分?jǐn)?shù))Tween-20的Tris-HCl緩沖液(TBST)洗滌，與辣根過氧化物酶標(biāo)記的二抗在室溫下孵育2 h。再經(jīng)TBST洗滌后，加入ECL化學(xué)發(fā)光試劑(美國Thermo Fisher公司)進(jìn)行膠片曝光顯影。

1.4 質(zhì)粒構(gòu)建

熒光素酶報(bào)告基因質(zhì)粒pGL3/APP的構(gòu)建：通過分析APP啟動(dòng)子區(qū)的評(píng)分較高的CRE元件的位置，筆者構(gòu)建了3種不同長(zhǎng)度的APP啟動(dòng)子熒光素酶報(bào)告基因質(zhì)粒pGL3/APP，其中APP1含有順式作用元件CRE 1～4，APP2含有CRE 3～4，APP3只含有CRE4。

1.5 聚合酶鏈?zhǔn)椒磻?yīng)(PCR)

以人cDNA為模板進(jìn)行PCR擴(kuò)增反應(yīng)，APP所用的引物為：上游，5′-CAC TTT GTG ATT CCC TAC CGC-3′，下游，5′-CAC CAG ACA TCC GAG TCA TCC-3′；GAPDH所用的引物為：上游，5′-TCA ACG GAT TTG GTC GTA TT-3′，下游，5′-CTG TGG TCA TGA GTC CTT CC-3′。各APP啟動(dòng)子區(qū)的引物分別是：APP1上游引物，5′-GGG GTA CCT GGG CCT CCT AAA GTG CTG-3′；APP2上游引物，5′-GGG GTA CCA GGC ACC CTT GTC AGC G-3′；APP3上游引物，5′-GGG GTA CCA ACC CAA GCC CAG AAC C-3′；所有的下游引物均為5′-GAA GAT CTA GGG CTG GGC CGA AAG-3′。反應(yīng)條件設(shè)定為：95 ℃ 3 min；95 ℃ 30 s，60 ℃ 30 s，72 ℃ 30 s，35個(gè)循環(huán)；72 ℃ 5 min。PCR產(chǎn)物用1.5%(質(zhì)量分?jǐn)?shù))瓊脂糖凝膠電泳分離。

1.6 細(xì)胞轉(zhuǎn)染

轉(zhuǎn)染前一天將HEK-293FT細(xì)胞以50%密度接種于細(xì)胞培養(yǎng)板，轉(zhuǎn)染當(dāng)天待細(xì)胞密度約80%左右，按照Lipofectamine 3000(Invitrogen公司，美國)說明書進(jìn)行操作，將熒光素酶報(bào)告基因質(zhì)粒pGL3/APP轉(zhuǎn)染入細(xì)胞。

1.7 雙熒光素酶報(bào)告基因系統(tǒng)分析

根據(jù)雙熒光素酶報(bào)告基因檢測(cè)系統(tǒng)(購自美國Promega公司)說明書進(jìn)行操作，測(cè)定熒光素酶激發(fā)所釋放的熒光值(A1)，樣品中內(nèi)參質(zhì)粒(pRL-TL)所攜帶的海腎熒光素酶激發(fā)底物所釋放熒光值(A2)。每一個(gè)樣品的A1/A2比值即為該質(zhì)粒轉(zhuǎn)染細(xì)胞后所表達(dá)的熒光素酶的相對(duì)活性。

1.8 統(tǒng)計(jì)學(xué)方法

2 結(jié)果

2.1 TSG對(duì)神經(jīng)細(xì)胞內(nèi)APP表達(dá)的影響

TSG(100 μmol/L)與人神經(jīng)母細(xì)胞瘤細(xì)胞系SH-SY5Y細(xì)胞孵育48 h，然后應(yīng)用Western blotting和RT-PCR方法檢測(cè)內(nèi)源性APP蛋白表達(dá)量的變化，結(jié)果顯示無論在蛋白水平還是mRNA水平，TSG可明顯降低神經(jīng)細(xì)胞內(nèi)APP的表達(dá)水平(圖1A，1.000±0.043vs0.612±0.044,P<0.001；圖1B,1.000±0.032vs0.750±0.047,P<0.01)。

圖1 TSG對(duì)SH-SY5Y細(xì)胞內(nèi)APP表達(dá)的影響Fig.1 Effect of TSG on APP expression in SH-SY5Y cells

SH-SY5Y cells were incubated with 100 μmol/L TSG for 48 h.The endogenous APP level was determined by Western blotting (A) and RT-PCR (B),and GAPDH was used as a reference.Data were presented as mean ±SE,n=4.**P<0.01***P<0.001vscontrol group;TSG:tetrahydroxystilbene glucoside;APP:amyloid precursor protein.

2.2 CREB對(duì)SH-SY5Y細(xì)胞內(nèi)APP表達(dá)的影響

筆者在上述研究中發(fā)現(xiàn)，TSG對(duì)SH-SY5Y細(xì)胞內(nèi)CREB磷酸化水平有一定升高作用(圖2A)，提示其對(duì)CREB有一定激活作用。為了進(jìn)一步研究CREB對(duì)APP表達(dá)的影響，筆者用PKA激活劑Forskolin(10 μmol/L)與SH-SY5Y細(xì)胞孵育12 h，以此來活化CREB。通過Western blotting分析，筆者檢測(cè)到磷酸化CREB(p-CREB)表達(dá)明顯升高(圖2B，1.000±0.042vs1.423±0.063,P<0.01)，表明CREB被激活，同時(shí)發(fā)現(xiàn)APP表達(dá)明顯降低(圖2B，1.000±0.057vs0.684±0.043,P<0.01)。這些結(jié)果提示CREB通過與CRE元件結(jié)合，對(duì)APP的表達(dá)有下調(diào)作用。

2.3 APP啟動(dòng)子區(qū)CRE元件分析及雙熒光素酶報(bào)告基因質(zhì)粒的構(gòu)建

為了研究人APP的表達(dá)調(diào)控，筆者由轉(zhuǎn)錄起始點(diǎn)開始，分析APP轉(zhuǎn)錄起始位點(diǎn)上游2 000 bp左右的啟動(dòng)子區(qū)，用Matinspector軟件分析了該區(qū)域的CRE元件，篩選出4個(gè)評(píng)分較高的CRE元件，分別位于 -1 972～-1 952(CRE1)，-1 886～-1 866(CRE2)，-1 031～-1 011(CRE3)，-711～-691(CRE4)(圖3A)。按照其分布，筆者構(gòu)建了3個(gè)不同長(zhǎng)度的APP啟動(dòng)子截?cái)囿w，命名為APP 1～3，其中APP 1含有順式作用元件CRE 1～4；APP 2含有CRE 3～4；APP3只含有CRE4(圖3B)。然后將它們分別連接到pGL3載體中，對(duì)pGL3/APP質(zhì)粒進(jìn)行酶切鑒定，瓊脂糖凝膠電泳結(jié)果顯示酶切的條帶長(zhǎng)度均符合預(yù)期(圖3C)，進(jìn)一步的測(cè)序鑒定結(jié)果也提示熒光素酶報(bào)告基因質(zhì)粒構(gòu)建成功。

圖2 CREB對(duì)SH-SY5Y細(xì)胞內(nèi)APP表達(dá)的影響Fig.2 Effect of CREB on APP expression in SH-SY5Y cells

A:SH-SY5Y cells were incubated with 100 μmol/L TSG for 48 h.The endogenous CREB，phosphorylated CREB (p-CREB) and GAPDH protein levels were determined by Western blotting.The data are presented as mean ±SE,n=4.*P<0.05vscontrol group;B: SH-SY5Y cells were incubated with 10 μmol/L Forskolin (a PKA activator) for 12 h.The endogenous p-CREB,CREB,APP and GAPDH levels were determined by Western blotting assay.Quantitative data are presented as mean ±SE,n=4.**P<0.01vscontrol group;CREB:cAMP responsive element-binding protein;APP:amyloid precursor protein.

圖3 APP啟動(dòng)子區(qū)CRE元件的分析及雙熒光素酶報(bào)告基因質(zhì)粒的構(gòu)建Fig.3 Analysis of CRE elements in APP promoter and construction of luciferase double reporter gene plasmids

A：The promoter of human APP gene was analyzed by matinspector software.There were four highly ranked CRE elements named as CRE 1 to 4 listed in the panel；B：According to their positions,we generated three deletion mutant forms of APP promoter；C：The APP promoter deletion mutants was amplified by PCR and cloned into pGL3 vectors.The plasmids were digested by restriction enzymes and subjected for agarose gel electrophoresis；APP:amyloid precursor protein；CRE：cAMP responsive element.

2.4 TSG對(duì)APP啟動(dòng)子質(zhì)粒轉(zhuǎn)染細(xì)胞中APP雙熒光素酶報(bào)告基因表達(dá)的影響

將pGL3/APP1～3質(zhì)粒分別和pRL-TK質(zhì)粒(作為內(nèi)參)共同轉(zhuǎn)染至HEK-293FT細(xì)胞中，48 h后用熒光雙報(bào)告基因檢測(cè)系統(tǒng)分析APP啟動(dòng)子區(qū)各截?cái)囿w中熒光素酶表達(dá)的情況。結(jié)果顯示，與對(duì)照組(pGL3空載體)相比，pGL3/APP1～3的熒光素酶表達(dá)量有不同程度的增高，其中含有CRE數(shù)目最多的APP1熒光素酶表達(dá)最高，表明含有CRE的APP啟動(dòng)子區(qū)可以很好地驅(qū)動(dòng)熒光素酶基因的表達(dá)，并且與CRE的量呈正相關(guān)(圖4)。TSG(100 μmol/L)與pGL3/APP1～3轉(zhuǎn)染細(xì)胞孵育48 h后，APP啟動(dòng)子區(qū)驅(qū)動(dòng)熒光素酶表達(dá)的能力有所下降，尤其是TSG對(duì)APP1組的抑制作用最為明顯，與未處理組相比差異有統(tǒng)計(jì)學(xué)意義(235.5±2.50vs177.0 ±4.00,P<0.01；圖4),而TSG對(duì)APP其他兩個(gè)截?cái)囿w2和3無顯著影響(P=0.21，P=0.11)，結(jié)果提示TSG可能影響CREB與CRE1和CRE2元件的結(jié)合。

圖4 TSG對(duì)APP啟動(dòng)子質(zhì)粒轉(zhuǎn)染細(xì)胞中APP雙熒光素酶報(bào)告基因表達(dá)的影響Fig.4 Effect of TSG on luciferase double reporter gene expression of APP in APP promoter plamid-transfected cells

The pGL3/APP or pGL3-basic vectors were transfected into HEK-293FT cells together with pRL-TK (as a reference).The drug treated groups were incubated with 100 μmol/L TSG since transfection.After 48 h,the cells were lysed,and luciferase activity assay was measured by luciferase double reporter gene system.Data were presented as mean ±SE,n=4.**P<0.01vsthe non-TSG treated pGL3/APP1 group.

3 討論

二苯乙烯苷(TSG)是中藥何首烏的主要有效成分。筆者以前的研究[10]結(jié)果顯示，TSG十二指腸給藥可在家兔血漿和腦脊液中檢測(cè)到，表明TSG能夠透過血-腦脊液屏障，這為TSG防治AD提供了可行性。本課題組自行研制的中藥5類新藥泰思膠囊(二苯乙烯苷)獲得國家食品藥品監(jiān)督管理總局(China Food and Drug Administration,CFDA)臨床研究批件，已完成治療AD的Ⅱ期臨床試驗(yàn)，證明有效安全，目前正在進(jìn)行Ⅲ期臨床試驗(yàn)。在臨床前藥效學(xué)研究中，本課題組在應(yīng)用APP轉(zhuǎn)基因小鼠模型的實(shí)驗(yàn)中發(fā)現(xiàn)，TSG灌胃給藥能夠改善模型鼠的學(xué)習(xí)記憶能力，減少腦顳葉皮質(zhì)淀粉樣斑塊的數(shù)目和面積，減低皮質(zhì)和海馬Aβ量；在體外實(shí)驗(yàn)中，TSG能明顯抑制β分泌酶活性[7-9]。這些前期研究結(jié)果提示TSG與APP-Aβ通路關(guān)系密切。因此，在本實(shí)驗(yàn)中筆者研究了TSG對(duì)神經(jīng)細(xì)胞內(nèi)APP表達(dá)的影響及其作用機(jī)制，發(fā)現(xiàn)TSG顯著抑制神經(jīng)細(xì)胞內(nèi)APP表達(dá)；進(jìn)一步通過克隆APP啟動(dòng)子區(qū)熒光素酶報(bào)告基因質(zhì)粒，發(fā)現(xiàn)TSG可能通過影響CREB與CRE元件的結(jié)合，從而下調(diào)APP的表達(dá)。

CREB通過結(jié)合啟動(dòng)子從而調(diào)節(jié)其下游基因的轉(zhuǎn)錄。CREB活性受其自身絲氨酸133位點(diǎn)磷酸化調(diào)控，該位點(diǎn)的磷酸化是由蛋白激酶A(protein kinase A，PKA)調(diào)控的[11]。CREB在神經(jīng)系統(tǒng)的作用至關(guān)重要，可參與學(xué)習(xí)記憶、細(xì)胞周期調(diào)控、神經(jīng)元誘導(dǎo)分化等生理活動(dòng)，還影響神經(jīng)退行性疾病的發(fā)生發(fā)展[12-13]。作為細(xì)胞內(nèi)的重要轉(zhuǎn)錄因子之一，已有研究[14-16]報(bào)道CREB調(diào)節(jié)與AD發(fā)病相關(guān)的多個(gè)基因的表達(dá)。由此提示，在AD的發(fā)病過程中，CREB通路活性的改變可能影響AD患者病理的進(jìn)展。

本研究中，筆者發(fā)現(xiàn)隨著APP啟動(dòng)子區(qū)CRE數(shù)目的減少，熒光素酶質(zhì)粒的熒光強(qiáng)度也隨之下降，說明CRE元件對(duì)于APP表達(dá)至關(guān)重要。此外，TSG處理對(duì)于APP1的影響明顯大于其他兩個(gè)截?cái)囿w，這提示TSG對(duì)CRE1和CRE2的影響對(duì)APP表達(dá)更為重要，筆者推測(cè)TSG可能通過調(diào)節(jié)CREB與CRE元件的結(jié)合從而下調(diào)APP表達(dá)，這也將是筆者下一步需要探討和證實(shí)的問題。

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EffectsoftetrahydroxystilbeneglucosideonamyloidprecursorproteinexpressioninnervecellsanditsmechanismrelatedtoCREBregulation

Yin Xiaomin1,2,Xu Ting2,Dai Wufei2,Qian Jiayi2,Chen Chen1,Zhang Lan1,Li Lin1*

(1.DepartmentofPharmacology,XuanwuHospital,CapitalMedicalUniversity,BeijingEngineeringResearchCenterforNerveSystemDrugs,Beijing100053,China;2.DepartmentofBiochemistry,MedicalSchoolofNantongUniversity,Nantong226001,JiangsuProvince,China)

ObjectiveTo explore the effects of tetrahydroxystilbene glucoside (TSG) on the expression of amyloid precursor protein (APP) and its mechanism in cultured nerve cells.MethodsThe expression of APP and cAMP responsive element-binding protein (CREB) was detected by Western blotting assay.The CRE cis-elements of APP promoter were analyzed by Matinspector software.The plasmids expressing serial deletion mutants of APP promoter were constructed.The luciferase activity in cells was measured by luciferase double reporter gene system.ResultsActivation of CREB by Forskolin down-regulated APP expression in SH-SY5Y cells.The luciferase expression plasmids of APP promoter containing CRE elements were successfully constructed.TSG treatment decreased APP level in SH-SY5Y cells,and reduced luciferase activity of APP reporter gene containing CRE1 and CRE2 cis-elements in APP promoter plasmids-transfected cells.ConclusionTSG down-regulated APP expression in nerve cells,and its mechanism may be related to affecting the binding of CREB with CRE cis-elements.

tetrahydroxystilbene glucoside;amyloid precursor protein;cAMP responsive element-binding protein;Alzheimer’s disease

國家自然科學(xué)基金(81273498,81473373,81503077)，國家重大新藥創(chuàng)制重大專項(xiàng)(2015ZX09101016001)，江蘇省大學(xué)生創(chuàng)新訓(xùn)練計(jì)劃項(xiàng)目(201610304095x)。This study was supported by National Natural Science Foundation of China (81273498,81473373,81503077),National Science and Technology Major Project for New Drug Research and Development of China (2015ZX09101-016),Jiangsu Students’ Innovation and Entrepreneurship Training Program (201610304095x).

*Corresponding author，E-mail：linlixw@126.com

時(shí)間：2017-12-13 21∶25

http://kns.cnki.net/kcms/detail/11.3662.R.20171213.2125.054.html

10.3969/j.issn.1006-7795.2017.06.012]

R749.1

2017-10-01)

編輯 陳瑞芳