IGF-1對(duì)Aβ25-35誘導(dǎo)PC12細(xì)胞凋亡的保護(hù)作用及其機(jī)制

陸蔚天,孫善全,許士葉,黃 娟*

(1重慶醫(yī)科大學(xué)神經(jīng)科學(xué)研究中心,重慶 400016;2重慶醫(yī)科大學(xué)人體解剖學(xué)教研室;*通訊作者,E-mail:huangjuan@cqmu.edu.cn)

IGF-1對(duì)Aβ25-35誘導(dǎo)PC12細(xì)胞凋亡的保護(hù)作用及其機(jī)制

陸蔚天1,2,孫善全1,2,許士葉1,2,黃 娟1,2*

(1重慶醫(yī)科大學(xué)神經(jīng)科學(xué)研究中心,重慶 400016;2重慶醫(yī)科大學(xué)人體解剖學(xué)教研室;*通訊作者,E-mail:huangjuan@cqmu.edu.cn)

目的 探討胰島素樣生長(zhǎng)因子-1(insulin-like growth factor-1,IGF-1)對(duì)β-淀粉樣蛋白25-35(amyloid-beta protein,Aβ25-35)誘導(dǎo)PC12細(xì)胞凋亡的保護(hù)作用及可能的信號(hào)機(jī)制。 方法 以10 μmol/L Aβ25-35處理PC12細(xì)胞作為阿爾茨海默病(Alzheimer’s disease,AD)細(xì)胞模型。實(shí)驗(yàn)分為對(duì)照組、Aβ25-35處理組、IGF-1+Aβ25-35處理組。條件孵育24 h,采用噻唑藍(lán)比色法(methyl thiazolyltetrazolium,MTT)檢測(cè)細(xì)胞存活率,Western blot檢測(cè)各組細(xì)胞磷酸化-Akt(phospho-Akt,p-Akt)蛋白表達(dá)水平。 結(jié)果 MTT法檢測(cè)顯示,10 μmol/L Aβ25-35孵育24 h,PC12細(xì)胞存活率為(39.8±3.7)%,與對(duì)照組[(100.0±2.3)%]相比顯著下降(P<0.05)。IGF-1+Aβ25-35處理組,PC12細(xì)胞存活率為(56.4±4.2)%,與Aβ25-35處理組[(39.8±3.7)%]相比顯著增高(P<0.05)。Western blot顯示,IGF-1+Aβ25-35處理組p-Akt蛋白表達(dá)水平(0.794±0.037)高于Aβ25-35處理組(0.493±0.050),差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)。 結(jié)論 IGF-1能抑制Aβ25-35所導(dǎo)致的PC12細(xì)胞凋亡,其保護(hù)作用可能與上調(diào)Akt磷酸化的表達(dá)有關(guān)。

胰島素樣生長(zhǎng)因子1; 淀粉樣蛋白; 細(xì)胞凋亡; PI3K-Akt信號(hào)通路

阿爾茨海默病(Alzheimer’s disease,AD)是一種常見的漸進(jìn)性、不可逆的神經(jīng)系統(tǒng)退行性疾病。由淀粉樣蛋白(amyloid-beta protein,Aβ)聚集在神經(jīng)元外形成的老年斑是AD最明顯的病理特征之一。已有的大量研究表明,Aβ可導(dǎo)致AD患者腦內(nèi)出現(xiàn)氧化應(yīng)激、線粒體功能障礙、神經(jīng)元凋亡等諸多不良后果[1,2]。淀粉樣蛋白25-35位的氨基酸序列(Aβ25-35)是Aβ產(chǎn)生神經(jīng)毒性的必要結(jié)構(gòu)[3]。用Aβ25-35處理細(xì)胞或者動(dòng)物,可誘導(dǎo)細(xì)胞產(chǎn)生氧化應(yīng)激、炎癥反應(yīng)、誘導(dǎo)細(xì)胞凋亡,并引起動(dòng)物出現(xiàn)認(rèn)知功能障礙等AD特征性表現(xiàn)[4,5]。因此,闡明Aβ對(duì)AD神經(jīng)毒性的發(fā)生機(jī)制,并探尋有效應(yīng)對(duì)Aβ毒性作用的可靠對(duì)策,成為預(yù)防和治療AD的關(guān)鍵所在。胰島素樣生長(zhǎng)因子(insulin-like growth factor 1,IGF-1)是胰島素家族的成員之一,與胰島素在分子結(jié)構(gòu)和功能上具有高度同源性。在中樞神經(jīng)系統(tǒng)內(nèi),IGF-1作為重要的神經(jīng)調(diào)節(jié)因子,參與調(diào)節(jié)細(xì)胞發(fā)育存活、能量代謝和學(xué)習(xí)記憶等重要生理過程[6]。已有研究表明,IGF-1可以通過加快清除腦內(nèi)Aβ,緩解記憶障礙等,從而對(duì)AD發(fā)揮保護(hù)作用[7-9]。但是,目前對(duì)于IGF-1通過哪些信號(hào)通路發(fā)揮AD細(xì)胞保護(hù)作用仍在研究之中。因此,本實(shí)驗(yàn)以Aβ25-35處理PC12細(xì)胞制作AD細(xì)胞模型,并探討IGF-1對(duì)Aβ25-35所致PC12細(xì)胞凋亡的保護(hù)作用及潛在作用機(jī)制。

1 材料和方法

1.1 細(xì)胞株及主要試劑

1.2 細(xì)胞培養(yǎng)

細(xì)胞培養(yǎng)于含10%胎牛血清的DMEM/F12培養(yǎng)基,在37 ℃、5%CO2培養(yǎng)箱中培養(yǎng),每2-3 d傳代1次。取對(duì)數(shù)生長(zhǎng)期細(xì)胞進(jìn)行實(shí)驗(yàn)。實(shí)驗(yàn)分為3組:對(duì)照組、Aβ25-35組(加入終濃度為10 μmol/L的Aβ25-35處理)、IGF-1+Aβ25-35組(加入終濃度為100 ng/ml的IGF-1和10 μmol/L的Aβ25-35共同處理)。條件培養(yǎng)24 h,進(jìn)行后續(xù)檢測(cè)。

1.3 MTT法檢測(cè)細(xì)胞存活率

將細(xì)胞按1×105個(gè)/ml濃度接種于96孔板,每孔100 μl,對(duì)照組加入等量培養(yǎng)液,每組設(shè)5個(gè)復(fù)孔。終止培養(yǎng)前4 h每孔加入5 mg/ml的MTT10 μl,置于培養(yǎng)箱37 ℃孵育4 h。去除上清,每孔加入150 μl DMSO,充分吹打甲臜結(jié)晶后置于搖床混勻10 min,用酶標(biāo)儀檢(BIO-RAD Model)檢測(cè)每孔細(xì)胞A450 nm處吸光度值。以對(duì)照組吸光度為100%,其余實(shí)驗(yàn)組細(xì)胞存活率(%)=A實(shí)驗(yàn)組/A對(duì)照組×100%。

1.4 Western blot檢測(cè)蛋白表達(dá)

棄去培養(yǎng)液,用4 ℃的PBS洗滌細(xì)胞2次。加入Western細(xì)胞裂解液吹打細(xì)胞。收集細(xì)胞,10 000×g離心5 min。采用BCA法進(jìn)行蛋白定量。取等量蛋白(20 μg)進(jìn)行10%的聚丙烯酰氨凝膠電泳(SDS-PAGE),100 V恒壓電泳1 h,350 mA轉(zhuǎn)膜56 min,5%脫脂奶粉封閉30 min,加入小鼠抗大鼠一抗(1 ∶1 000)4 ℃過夜,PBST洗3次,每次10 min;加入羊抗小鼠二抗(1 ∶1 000)37 ℃孵育60 min,PBST洗3次,每次10 min。加入辣根過氧化物酶標(biāo)記的DAB顯色。用Quantity one軟件測(cè)定各組目的蛋白條帶的灰度值,用p-Akt/Akt條帶比值作為蛋白相對(duì)值。

1.5 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 IGF-1抑制Aβ25-35對(duì)PC12細(xì)胞誘導(dǎo)的細(xì)胞凋亡

用10 μmol/L的Aβ25-35孵育24 h,PC12細(xì)胞存活率為(39.8±3.7)%,與對(duì)照組[(100.0±2.3)%]相比顯著下降(P<0.05)。同時(shí)給予100 ng/ml IGF-1和10 μmol/L Aβ25-35處理后,細(xì)胞存活率為(56.4±4.2)%，與單獨(dú)Aβ25-35處理組[(39.8±3.7)%]相比,細(xì)胞存活率升高(P<0.05,見圖1)。

與對(duì)照組相比,*P<0.05;與Aβ25-35組相比,#P<0.05圖1 IGF-1抑制Aβ25-35對(duì)PC12細(xì)胞誘導(dǎo)的細(xì)胞凋亡Figure 1 IGF-1 inhibited the Aβ25-35-induced apoptosis of PC12 cells

2.2 IGF-1促進(jìn)Aβ25-35所致PC12細(xì)胞損傷模型中Akt的磷酸化

對(duì)照組PC12細(xì)胞p-Akt表達(dá)水平較低(0.307±0.037),Aβ25-35處理后,PC12細(xì)胞p-Akt相對(duì)表達(dá)量升高(0.493±0.050),差異具有統(tǒng)計(jì)學(xué)意義(P<0.05,見圖2)。IGF-1+Aβ25-35組PC12細(xì)胞p-Akt表達(dá)水平最高(0.794±0.037),與Aβ25-35組相比,差異具有統(tǒng)計(jì)學(xué)意義(P<0.05,見圖2)。

與對(duì)照組相比,*P<0.05;與Aβ25-35組相比,#P<0.05圖2 免疫印跡檢測(cè)p-Akt蛋白在各組中的表達(dá)Figure 2 Expression of p-Akt protein in PC12 cells by Western blot

3 討論

在眾多導(dǎo)致AD的因素中,Aβ聚集形成淀粉樣變性(amyloidosis)是目前公認(rèn)的最為關(guān)鍵的一個(gè)致病因素。Aβ可以引起AD患者腦內(nèi)突觸功能障礙、神經(jīng)炎性反應(yīng)、氧化應(yīng)激、神經(jīng)元丟失和認(rèn)知障礙等后果。AD患者腦內(nèi)Aβ過度聚集并且在神經(jīng)元外沉積形成老年斑,是AD特征性標(biāo)志之一[10]。本研究采用淀粉樣蛋白神經(jīng)毒性片段Aβ25-35處理PC12細(xì)胞建立AD細(xì)胞模型,并用IGF-1干預(yù),以探討IGF-1對(duì)AD的保護(hù)作用及可能機(jī)制。本實(shí)驗(yàn)中,加入10 μmol/L的Aβ25-35處理后,與對(duì)照組相比,PC12細(xì)胞存活率顯著下降,結(jié)果證實(shí)Aβ25-35對(duì)PC12細(xì)胞生長(zhǎng)具有抑制作用,采用Aβ25-35處理可作為AD的細(xì)胞模型。

在腦內(nèi),IGF-1通過廣泛表達(dá)于大、小鼠皮質(zhì)、海馬、間腦和嗅球等部位的IGF-1受體發(fā)揮作用[8]。研究顯示,IGF-1能夠促進(jìn)神經(jīng)細(xì)胞的增殖及存活,對(duì)細(xì)胞具有保護(hù)作用[11,12]。本研究中,加入IGF-1處理可以顯著減少Aβ25-35對(duì)PC12細(xì)胞造成的細(xì)胞凋亡,結(jié)果提示IGF-1對(duì)PC12細(xì)胞發(fā)揮保護(hù)作用。研究顯示IGF-1通過與IGF-1受體結(jié)合后可激活磷脂酰肌醇3-羥基激酶(phosphatidylinositol 3-kinase,PI3K)-絲氨酸/蘇氨酸蛋白激酶Akt等下游信號(hào)通路以發(fā)揮神經(jīng)保護(hù)作用。PI3K-Akt組成的信號(hào)通路是細(xì)胞內(nèi)的一條重要信號(hào)通路,可以調(diào)節(jié)與細(xì)胞生長(zhǎng)、分化與遷移及凋亡等重要過程中基因的表達(dá),并啟動(dòng)下游級(jí)聯(lián)系統(tǒng)[12]。研究顯示,IGF-1可以激活PI3K信號(hào)通路,從而磷酸化目的分子Akt,磷酸化的Akt激活或抑制下游靶蛋白,如葡萄糖合成激酶-3(glycogen synthase kinase-3,GSK-3β)、雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、Bad、caspase-9等,參與介導(dǎo)葡萄糖轉(zhuǎn)運(yùn)、脂肪和蛋白合成,進(jìn)而調(diào)控細(xì)胞存活與分化、血管生成和能量代謝等重要過程[13-15]。

已有研究提示,PI3K-Akt信號(hào)通路可能通過以下多種機(jī)制參與調(diào)控細(xì)胞凋亡:①抑制Forkhead box O磷酸化及向細(xì)胞核的轉(zhuǎn)移,從而阻止FOXO調(diào)控促凋亡基因的轉(zhuǎn)錄[15]。②Akt磷酸化后抑制促凋亡蛋白家族成員Bad的活性[16]。③可通過阻止Bax轉(zhuǎn)移至線粒體,以減少細(xì)胞色素C的釋放,從而達(dá)到細(xì)胞保護(hù)的作用[17]。④該通路可抑制GSK-3β活性,抑制糖原合成,減少具有神經(jīng)毒性tau的磷酸化,從而發(fā)揮神經(jīng)保護(hù)作用[18]。現(xiàn)有研究表明,體內(nèi)多種分子如表皮生長(zhǎng)因子、IGF-1R等可通過刺激PI3K-Akt信號(hào)通路來調(diào)節(jié)神經(jīng)元的存活及死亡。研究顯示,若細(xì)胞內(nèi)、外因素刺激干擾IGF-1信號(hào),可引起PI3K通路轉(zhuǎn)導(dǎo)紊亂,導(dǎo)致出現(xiàn)Aβ沉積、tau過度磷酸化、氧化應(yīng)激,甚至神經(jīng)元凋亡等不良后果[18]。本研究結(jié)果顯示IGF-1處理可增加PC12細(xì)胞p-Akt的表達(dá),同時(shí)顯著抑制Aβ25-35誘導(dǎo)的PC12細(xì)胞凋亡。據(jù)此推測(cè),IGF-1通過激活PI3K-Akt信號(hào)通路,從而發(fā)揮對(duì)PC12細(xì)胞的保護(hù)作用。

綜上所述,本研究結(jié)果顯示IGF-1可抑制Aβ25-35誘導(dǎo)PC12細(xì)胞發(fā)生凋亡,其保護(hù)作用可能與激活PI3K-Akt信號(hào)通路有關(guān)。AD發(fā)生發(fā)展過程中,患者腦內(nèi)IGF-1水平及PI3K-Akt信號(hào)通路異常與神經(jīng)元的變性丟失緊密相關(guān)。本研究結(jié)果為進(jìn)一步開展AD的藥物研發(fā)提供了實(shí)驗(yàn)數(shù)據(jù)。

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ProtectiveeffectofIGF-1onAβ25-35-inducedapoptosisofPC12cellsanditsmechanism

LU Weitian1,2,SUN Shanquan1,2,XU Shiye1,2,HUANG Juan1,2*

(1InstituteofNeuroscience,ChongqingMedicalUniversity,Chongqing400016,China;2DepartmentofHumanAnatomy,ChongqingMedicalUniversity;*Correspondingauthor,E-mail:huangjuan@cqmu.edu.cn)

ObjectiveTo investigate the protective effect of insulin-like growth factor 1(IGF-1) on amyloid-beta protein 25-35 (Aβ25-35)-induced neurotoxicity in PC12 cells and its possible mechanisms.MethodsPC12 cells were treated with 10 μmol/L Aβ25-35to establish the cell model of Alzheimer’s disease (AD). PC12 cells were divided into three groups: control group, Aβ25-35group and IGF-1+Aβ25-35group. After 24 h treatment, the cell viability was detected by MTT assay. The expression of phospho-Akt protein was detected by Western blot.ResultsCompared with control group, the viability in Aβ25-35group was significantly decreased[(100.0±2.3)%vs(39.8±3.7) %,P<0.05]. Meanwhile, the cell viability in IGF-1+Aβ25-35group was significantly higher than that in Aβ25-35group [(56.4±4.2)%vs(39.8±3.7)%,P<0.05]. Western blot results showed that the expression of phospho-Akt in IGF-1+Aβ25-35group(0.794±0.037)was higher than that in Aβ25-35group(0.493±0.050,P<0.05).ConclusionIGF-1 may protect PC12 cells against Aβ25-35-induced apoptosis by up-regulating the expression of phospho-Akt.

insulin-like growth factor 1; amyloid-β protein; apoptosis; PI3K-Akt signaling pathway

R363

A

1007-6611(2017)09-0900-04

10.13753/j.issn.1007-6611.2017.09.007

國(guó)家自然科學(xué)基金資助項(xiàng)目(81601051);重慶市基礎(chǔ)與前沿研究計(jì)劃項(xiàng)目(cstc2016jcyjA0172);重慶市渝中區(qū)科技計(jì)劃項(xiàng)目(20140115);重慶市教委科學(xué)技術(shù)研究項(xiàng)目(KJ1600222);重慶醫(yī)科大學(xué)國(guó)家自然科學(xué)基金預(yù)研資助項(xiàng)目(NSFYY201525)

陸蔚天,男,1979-05生,博士,副教授,E-mail:weitianlu@cqmu.edu.cn

2017-06-08