芹菜素對(duì)血小板源生長(zhǎng)因子誘導(dǎo)血管平滑肌細(xì)胞遷移的影響*

關(guān)紅菁,崔昌萍,黃際友,淑芬

(1.武漢大學(xué)人民醫(yī)院心血管內(nèi)科,武漢 430060;2.武漢大學(xué)心血管病研究所,武漢 430060;3.解放軍75240部隊(duì)衛(wèi)生隊(duì),潮州 521000)

·藥物研究·

芹菜素對(duì)血小板源生長(zhǎng)因子誘導(dǎo)血管平滑肌細(xì)胞遷移的影響*

關(guān)紅菁1,2,崔昌萍1,2,黃際友3,淑芬1,2

(1.武漢大學(xué)人民醫(yī)院心血管內(nèi)科,武漢 430060;2.武漢大學(xué)心血管病研究所,武漢 430060;3.解放軍75240部隊(duì)衛(wèi)生隊(duì),潮州 521000)

目的 研究芹菜素在體外對(duì)血小板源生長(zhǎng)因子(PDGF)誘導(dǎo)的血管平滑肌細(xì)胞(VSMC)遷移作用的影響及可能的分子機(jī)制。方法酶消化法獲取大鼠胸主動(dòng)脈原代血管平滑肌細(xì)胞,Transwell小室方法評(píng)價(jià)芹菜素對(duì)PDGFBB誘導(dǎo)的VSMC遷移作用,Western blot方法檢測(cè)c-Jun N末端激酶(JNK)磷酸化水平。結(jié)果20 ng·mL-1PDGF-BB顯著促進(jìn)VSMC遷移,細(xì)胞數(shù)達(dá)到對(duì)照組的2.46倍,而12.5 μmol·L-1芹菜素預(yù)處理遷移細(xì)胞數(shù)僅為PDGF-BB組46.5%,不同劑量的芹菜素均能顯著抑制PDGF-BB誘導(dǎo)的VSMC遷移,12.5 μmol·L-1芹菜素預(yù)處理顯著抑制PDGF-BB對(duì)JNK磷酸化。結(jié)論芹菜素對(duì)PDGF-BB誘導(dǎo)的VSMC遷移有顯著的抑制作用,其機(jī)制可能部分與抑制JNK活化有關(guān)。

芹菜素;血小板源生長(zhǎng)因子;血管平滑肌細(xì)胞;細(xì)胞運(yùn)動(dòng)

動(dòng)脈粥樣硬化是多種心腦血管疾病的基礎(chǔ)病變。經(jīng)皮冠狀動(dòng)脈介入術(shù)(percutaneouscoronary intervention,PCI)的發(fā)展,極大改善動(dòng)脈粥樣硬化導(dǎo)致的血管阻塞,然而管腔內(nèi)再狹窄是阻礙這項(xiàng)技術(shù)獲得長(zhǎng)期成功的主要因素,血管平滑肌細(xì)胞(vascular smooth muscle cell,VSMC)的異常遷移是再狹窄過(guò)程中新生內(nèi)膜形成和管腔阻塞的重要因素之一[1-2]。血小板源生長(zhǎng)因子(platelet derived growth factor,PDGF)通過(guò)活化細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)啟動(dòng)多種生物效應(yīng)從而促進(jìn)平滑肌細(xì)胞遷移[3]。因此抑制PDGF誘導(dǎo)的VSMC遷移可能對(duì)抑制再狹窄的發(fā)生發(fā)揮重要作用。芹菜素屬于類黃酮中的黃酮亞類,通常存在于水果、蔬菜和茶葉中。研究表明芹菜素具有抗炎、抗氧化和抗腫瘤特性[4-5],然而芹菜素對(duì)于VSMC活化的作用尚未完全明確。筆者在本研究探討芹菜素對(duì)PDGF-BB誘導(dǎo)的VSMC遷移的作用以及可能的分子機(jī)制。

1 材料與方法

1.1 材料 芹菜素(批號(hào):10798,純度≥95%)購(gòu)自Sigma公司;PDGF-BB(批號(hào):Cyt-412)購(gòu)自Prospec公司;蛋白酶抑制藥Complete(批號(hào):04693124001),磷酸酶抑制劑PhosSTOP(批號(hào):04906837001),購(gòu)自Roche公司;DMEM/F12,胎牛血清購(gòu)自Gibco公司;抗總的和磷酸化的c-Jun N末端激酶(c-Jun N-terminalkinase,JNK)抗體(批號(hào):4668),抗甘油醛-3-磷酸脫氫酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)抗體(批號(hào):2118)購(gòu)自cell signaling technology公司,抗平滑肌α-肌動(dòng)蛋白(smooth muscle α-actin,SM αactin)抗體購(gòu)自Abcam公司(批號(hào):ab5694),Transwell系統(tǒng)購(gòu)自Corning公司。

1.2 血管平滑肌細(xì)胞的培養(yǎng)與鑒定 采用酶消化法[6]分離血管平滑肌細(xì)胞。無(wú)菌條件下取斯?jié)娎鄹瘛ざ嗬?Sprague-Dawley,SD)大鼠(體質(zhì)量100～150 g)胸主動(dòng)脈,清除血凝塊及周?chē)窘M織,將Ⅰ型膠原酶,Ⅰ型彈性蛋白酶和大豆胰蛋白酶抑制劑配制成混合消化液,37℃預(yù)消化胸主動(dòng)脈30 min,于體視顯微鏡下剝離血管外膜,將血管切成寬1 mm的血管環(huán),再于37℃消化2 h,離心收集細(xì)胞,細(xì)胞培養(yǎng)于含10%胎牛血清的DMEM/F12中。待生長(zhǎng)接近融合時(shí)傳代,血管平滑肌細(xì)胞的純度用細(xì)胞形態(tài)和VSMC SM α-actin染色鑒定。實(shí)驗(yàn)使用細(xì)胞為5～12代。

1.3 細(xì)胞遷移 VSMC的異常遷移對(duì)血管新生內(nèi)膜增生具有重要作用。芹菜素已被證實(shí)為有效的抑制細(xì)胞生長(zhǎng)的物質(zhì),然而芹菜素是否對(duì)VSMC的遷移有抑制作用仍有待研究。本研究用Transwell方法評(píng)價(jià)了芹菜素對(duì)VSMC遷移的作用。參考既往文獻(xiàn),選取兩個(gè)濃度的芹菜素進(jìn)行研究[7]。細(xì)胞分為4組,對(duì)照組,PDGF-BB組,12.5 μmol·L-1芹菜素組,25.0 μmol·L-1芹菜素組,芹菜素組分別用不同濃度的芹菜素預(yù)處理細(xì)胞1 h后,再用PDGF趨化細(xì)胞。VSMC 25 μmol·L-1以每孔5×104個(gè)(50 μL)的密度接種于上室,貼壁30 min,將25.0 μmol·L-1芹菜素50 μL加入上室,12.5 μmol·L-1芹菜素100 μL加入下室預(yù)處理細(xì)胞1 h,隨后PDGF-BB 20 ng·mL-1加入下室作為趨化劑。細(xì)胞遷移6 h后,用棉簽拭去膜上表面未遷移的細(xì)胞,遷移到下表面的細(xì)胞用多聚甲醛固定,隨后用0.1%結(jié)晶紫/20%甲醇溶液染色并計(jì)數(shù)。遷移細(xì)胞數(shù)目定義為每高倍視野遷移細(xì)胞數(shù)量。

1.4 Western blot VSMC接種于6 cm培養(yǎng)皿,細(xì)胞生長(zhǎng)至70%～80%融合后無(wú)血清饑餓24 h。12.5 μmol·L-1芹菜素預(yù)處理2 h后,予以PDGF-BB (20 ng·mL-1)刺激相應(yīng)的時(shí)間。細(xì)胞用預(yù)冷的磷酸鹽緩沖液(phosphate buffered solution,PBS)清洗3次,加入含有蛋白酶抑制劑和磷酸酶抑制劑的細(xì)胞裂解液裂解,14 500×g離心20 min,取上清液以BCA法測(cè)定蛋白質(zhì)含量。每個(gè)樣本取蛋白提取物20 μg用十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳分離,隨后轉(zhuǎn)印至Immobilon-FL(Millipore)膜上。含5%脫脂奶粉的洗膜緩沖液(TBST)封閉1 h,與不同的一抗4℃孵育過(guò)夜,之后與IRDye 800CW標(biāo)記的二抗于室溫下孵育1 h,Odyssey Imaging System獲取熒光信號(hào)。

1.5 統(tǒng)計(jì)學(xué)方法 采用SPSS13.0版統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(±s)表示,均經(jīng)正態(tài)性及方差齊性檢驗(yàn),多組間均數(shù)比較采用One Way ANOVA,兩組間均數(shù)比較采用LSD檢驗(yàn),P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 VSMC的鑒定 倒置顯微鏡下觀察細(xì)胞,可見(jiàn)單個(gè)VSMC為梭形,有多個(gè)細(xì)胞突起,細(xì)胞生長(zhǎng)致密時(shí)排列成束,部分重疊,呈特征性的“峰”“谷”狀生長(zhǎng)。運(yùn)用免疫熒光方法對(duì)VSMC行SM α-actin染色,熒光顯微鏡下可見(jiàn)細(xì)胞質(zhì)中與長(zhǎng)軸平行的條絲狀綠色熒光,即為肌動(dòng)蛋白,＞95%細(xì)胞染色呈陽(yáng)性(圖1)。

綠色:特異性陽(yáng)性染色;藍(lán)色:DAPI染色的細(xì)胞核;陽(yáng)性細(xì)胞超過(guò)95%圖1 熒光顯微鏡下觀察血管平滑肌細(xì)胞SM α-actin免疫熒光染色圖(×200)Green:specific positive staining;Blue:nucleus stained by DAPI;positive cells were more than 95%Fig.1 SM α-actin staining on vascular smooth muscle cells detected by fluorescence microscopy(×200)

2.2 芹菜素對(duì)PDGF-BB誘導(dǎo)的VSMC遷移的影響結(jié)果顯示,與對(duì)照組比較,PDGF-BB刺激6 h顯著促進(jìn)VSMC的遷移,遷移細(xì)胞數(shù)達(dá)到對(duì)照組的2.46倍,而12.5 μmol·L-1芹菜素預(yù)處理顯著抑制這一效應(yīng),遷移細(xì)胞數(shù)僅為PDGF-BB組46.5%。芹菜素的抑制作用呈現(xiàn)出劑量依賴的關(guān)系,25.0 μmol·L-1芹菜素即幾乎完全抑制PDGF促進(jìn)VSMC的遷移(圖2)。

2.3 芹菜素抑制VSMC遷移的分子機(jī)制 由于在細(xì)胞遷移實(shí)驗(yàn)中,12.5 μmol·L-1芹菜素預(yù)處理已經(jīng)顯示顯著抑制VSMC遷移,因此,只選取12.5 μmol·L-1芹菜素進(jìn)行分子機(jī)制的研究。PDGF-BB作用5 min后JNK即發(fā)生顯著磷酸化,而12.5 μmol·L-1芹菜素處理顯著抑制PDGF-BB對(duì)JNK的活化,PDGF-BB和芹菜素對(duì)JNK總蛋白水平均沒(méi)有影響,見(jiàn)圖3。

A.對(duì)照組;B.PDGF-BB組;C.12.5 μmol·L-1芹菜素組;D.25.0 μmol·L-1芹菜素組;與對(duì)照組比較,*1P＜0.05;與PDGF-BB組比較,*2P＜0.05圖2 芹菜素抑制PDGF-BB誘導(dǎo)的VSMC的遷移A.control group;B.PDGF-BB group;C.12.5 μmol·L-1apigenin group;D.25.0 μmol·L-1apigenin group;compared with control group,*1P＜0.05;compared with PDGF-BB group,*2P＜0.05Fig.2 Inhibition of Apigenin on migration of VSMC induced by PDGF-BB

與PDGF-BB組比較,*1P＜0.05圖3 芹菜素抑制PDGF-BB誘導(dǎo)的JNK磷酸化Compared with PDGF-BB group,*1P＜0.05Fig.3 Inhibition of Apigenin on JNK phosphorylation induced by PDGF-BB

3 討論

平滑肌細(xì)胞的遷移對(duì)于再狹窄的發(fā)展發(fā)揮重要作用。平滑肌細(xì)胞是新生內(nèi)膜主要的細(xì)胞成分,當(dāng)遷移到內(nèi)膜中,平滑肌細(xì)胞異常增殖并成為細(xì)胞外基質(zhì)蛋白的主要來(lái)源。因此尋找能抑制平滑肌細(xì)胞遷移的物質(zhì)有助于為再狹窄的治療提供新的策略。流行病學(xué)研究發(fā)現(xiàn)增加攝入蔬菜和水果有助于降低腫瘤和心血管疾病引起的死亡[8]。芹菜素是廣泛存在于蔬菜和水果中的植物化合物,研究發(fā)現(xiàn),芹菜素通過(guò)抗氧化作用,抑制細(xì)胞周期,促進(jìn)細(xì)胞凋亡等作用機(jī)制[9-10],對(duì)多種腫瘤細(xì)胞的生長(zhǎng)及侵襲具有顯著的抑制作用,但是芹菜素對(duì)于血管平滑肌細(xì)胞的作用目前尚未獲得一致結(jié)論[11-13]。因此本研究運(yùn)用Transwell細(xì)胞遷移檢測(cè)探討芹菜素對(duì)血管平滑肌細(xì)胞遷移的作用,發(fā)現(xiàn)預(yù)先用芹菜素處理顯著減少PDGF-BB誘導(dǎo)的細(xì)胞遷移,結(jié)果提示芹菜素是VSMC遷移的抑制劑,這個(gè)結(jié)果與LAMY等[12]研究結(jié)果一致。

芹菜素抑制血管平滑肌細(xì)胞遷移的分子機(jī)制目前尚不十分清楚。既往研究證實(shí)JNK的活化在多種細(xì)胞中都與細(xì)胞遷移增強(qiáng)相關(guān),因此在本研究中檢測(cè)了芹菜素對(duì)PDGF-BB誘導(dǎo)的JNK活化的作用。與既往研究一致,PDGF-BB誘導(dǎo)JNK快速的磷酸化。更重要的是,芹菜素干預(yù)導(dǎo)致JNK的磷酸化顯著減少,抑制血管平滑肌細(xì)胞JNK活化。ZHAN等[14]研究發(fā)現(xiàn),在平滑肌細(xì)胞中轉(zhuǎn)染突變失活的JNK導(dǎo)致PDGF-BB誘導(dǎo)的VSMC遷移減少約77%。與此同時(shí),KAVURMA等[15]也發(fā)現(xiàn)VSMC的遷移可以被JNK抑制劑SP600125所阻斷,這兩項(xiàng)研究提示了JNK活化在血管平滑肌細(xì)胞遷移中發(fā)揮了重要作用,因此芹菜素對(duì)于血管平滑肌細(xì)胞JNK活化的抑制可能是其對(duì)細(xì)胞遷移的抑制作用的機(jī)制之一。已有多項(xiàng)研究探討JNK參與細(xì)胞遷移調(diào)節(jié)的機(jī)制。研究顯示,粘著斑激酶可將整合素刺激信號(hào)傳導(dǎo)到JNK[16]。此外,細(xì)胞遷移的重要調(diào)節(jié)物Src也參與整合素到JNK的信號(hào)傳導(dǎo)[17]。由此可知,對(duì)于調(diào)控細(xì)胞運(yùn)動(dòng)的多種信號(hào)通路來(lái)說(shuō),JNK是共同的下游靶點(diǎn)。除了調(diào)控信號(hào)通路外,JNK還作用于細(xì)胞遷移的關(guān)鍵分子。細(xì)胞遷移需要細(xì)胞骨架重組,這個(gè)過(guò)程涉及到細(xì)胞骨架相關(guān)酪氨酸激酶的磷酸化以及粘著斑復(fù)合物的形成。paxillin是粘著斑銜接分子,研究發(fā)現(xiàn),paxillin的絲氨酸178位點(diǎn)可以被JNK磷酸化,當(dāng)這一位點(diǎn)發(fā)生突變而不能磷酸化時(shí),細(xì)胞運(yùn)動(dòng)顯著受限,結(jié)果提示這一過(guò)程對(duì)于快速細(xì)胞遷移是必要的[18]。然而在平滑肌細(xì)胞中,芹菜素抑制JNK活化后是否影響到以上信號(hào)通路仍需進(jìn)一步研究。

綜上所述,本研究證實(shí)芹菜素抑制PDGF-BB誘導(dǎo)的VSMC遷移,這一作用可能部分與其抑制JNK活化有關(guān)。研究提示芹菜素可能對(duì)再狹窄發(fā)生有一定的抑制作用,但這一作用仍需要通過(guò)動(dòng)物實(shí)驗(yàn)進(jìn)一步證實(shí)。

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DOI 10.3870/yydb.2014.10.003

Effects of Apigenin on Platelet Derived Growth Factor-induced Migration of Vascular Smooth Muscle Cells

GUAN Hong-jing1,2,CUI Chang-ping1,2,HUANG Ji-you3,SHU Fen1,2
(1.Department of Cardiology,Renmin Hospital of Wuhan University,Wuhan 430060,China;2.Cardiovascular Research Institute of Wuhan University, Wuhan 430060,China;3.Medical Unit of 75240 PLA Troops,Chaozhou 521000,China)

ObjectiveTo investigate the effects of apigenin on the migration of vascular smooth muscle cells(VSMC) induced by platelet derived growth factor(PDGF)-BB and the possible molecular mechanism.MethodsVSMCs were isolated from thoracic aortas of male Sprague-Dawley rats using enzyme digestion method.Migration of VSMCs was determined by transwell assay.Western blotting was carried out to evaluate phosphorylation of c-junN-terminal kinase(JNK).ResultsTreatment with PDGF-BB(20 ng·mL-1)significantly promote VSMC migration,the number of migrated cells was 2.46 times than that of control group.However,after 12.5 μmol·L-1apigenin pretreatment,the number of migrated cells was 46.5%of the PDGF-BB group.Various dose of apigenin can significantly inhibit VSMC migration induced by PDGF-BB,12.5 μmol·L-1apigenin treatment significantly inhibited PDGF-BB phosphorylation of JNK.ConclusionApigenin can suppress the migration of VSMC induced by PDGF-BB.These beneficial effects on VSMC were at least partly mediated by the inhibition of activity of JNK.

Apigenin;Platelet derived growth factor;Vascular smooth muscle cell;Cell movement

R965

A

1004-0781(2014)10-1265-04

2013-09-17

2014-04-18

*國(guó)家自然科學(xué)基金資助項(xiàng)目(81070089)

關(guān)紅菁(1973-),女,遼寧新賓人,副主任醫(yī)師,博士,主要從事心臟與血管的重構(gòu)研究。電話:027-88041911, E-mail:guanhj829@163.com。