鐘 毅,賴文巖,劉挺榕,郭志剛

(南方醫(yī)科大學(xué)南方醫(yī)院心內(nèi)科,廣州 510515)

血紅素氧合酶-1在姜黃素抑制氧化型低密度脂蛋白誘導(dǎo)大鼠血管平滑肌細(xì)胞增殖中的作用

鐘 毅,賴文巖,劉挺榕,郭志剛△

(南方醫(yī)科大學(xué)南方醫(yī)院心內(nèi)科,廣州 510515)

目的觀察姜黃素對(duì)氧化型低密度脂蛋白(Ox-LDL)誘導(dǎo)的大鼠血管平滑肌細(xì)胞(VSMC)增殖的影響,并探討血紅素氧合酶-1(HO-1)在姜黃素抑制血管平滑肌細(xì)胞增殖中的作用及機(jī)制?方法 體外分離培養(yǎng)大鼠血管平滑肌細(xì)胞并行免疫組化鑒定,以四甲基偶氮唑鹽(MTT)法測(cè)定細(xì)胞增殖率?逆轉(zhuǎn)錄-聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)檢測(cè)HO-1mRNA水平,蛋白免疫印跡法(Western blot)分別檢測(cè)HO-1及增殖細(xì)胞核抗原(PCNA)蛋白表達(dá)水平?結(jié)果Ox-LDL呈濃度依賴性(0～150μg/mL)地誘導(dǎo)VSMC增殖,達(dá)到200μg/mL具有細(xì)胞毒性;姜黃素呈濃度依賴性(40?80μmol/L)地抑制 Ox-LDL(100μg/mL)所誘導(dǎo)的VSMC增殖,HO-1抑制劑鋅原卟啉Ⅸ(ZnPPⅨ)可明顯減弱此抑制效應(yīng);姜黃素可誘導(dǎo)VSMC上調(diào)HO-1mRNA和蛋白表達(dá);姜黃素可減弱Ox-LDL誘導(dǎo)VSMCs增殖細(xì)胞核抗原的高表達(dá),此效應(yīng)可被HO-1抑制劑ZnPPⅨ抑制?結(jié)論姜黃素通過(guò)降低HO-1介導(dǎo)的PCNA表達(dá),進(jìn)而抑制Ox-LDL誘導(dǎo)大鼠血管平滑肌增殖?

姜黃素;氧合酶類;脂蛋白類,LDL

動(dòng)脈粥樣硬化(atherosclerosis,AS)的發(fā)生機(jī)制十分復(fù)雜,目前研究表明氧化型低密度脂蛋白(oxidized low density lipoprotein,Ox-LDL)及血管平滑肌細(xì)胞(vascular smooth muscle cell,VSMC)增殖是動(dòng)脈粥樣硬化斑塊形成的重要病理基礎(chǔ)[1]?姜黃素是從姜黃?郁金?莪術(shù)等植物中提取的一種生物多酚化合物,研究顯示姜黃素具有抗炎?抗氧化?促細(xì)胞凋亡和抑制細(xì)胞增殖等藥理作用,這些作用可有效拮抗AS的發(fā)生與發(fā)展?血紅素氧合酶-1(heme oxygenase-1,HO-1)是血紅素降解的起始酶和限速酶,發(fā)揮著抗炎?抗氧化?抑制細(xì)胞凋亡和改善組織微循環(huán)作用,動(dòng)物研究已經(jīng)證明過(guò)表達(dá)HO-1可以明顯延緩AS進(jìn)展[2],但是姜黃素抗AS的相關(guān)機(jī)制還不甚清楚?本研究旨在于細(xì)胞水平探討姜黃素對(duì)Ox-LDL誘導(dǎo)血管平滑肌細(xì)胞增殖的影響及其相關(guān)機(jī)制,為其藥理作用提供理論基礎(chǔ)?

1 材料與方法

1.1 材料 DMEM培養(yǎng)基?胎牛血清?胰蛋白酶購(gòu)自美國(guó)Gibico公司;姜黃素?鋅原卟啉Ⅸ(zine protoporphyrinⅨ,Zn-PPⅨ)購(gòu)自Sigma公司;Ox-LDL購(gòu)于北京普博斯生物科技有限公司;反轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)試劑均購(gòu)自美國(guó)Invitrogen公司;一抗?二抗均購(gòu)于美國(guó)Santa Cruz公司;SD大鼠購(gòu)自南方醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心;其余實(shí)驗(yàn)相關(guān)產(chǎn)品購(gòu)自碧云天生物技術(shù)公司?

1.2 方法

1.2.1 細(xì)胞培養(yǎng)與實(shí)驗(yàn)分組按常規(guī)組織貼塊方法[3]原代培養(yǎng)并傳代,取用第3～8代細(xì)胞?免疫組化法鑒定血管平滑肌細(xì)胞?血管平滑肌細(xì)胞用含10%胎牛血清的DMEM培養(yǎng)基于37℃?5%CO2下培養(yǎng)?實(shí)驗(yàn)分組:(1)正常對(duì)照組:不加任何處理;(2)不同終濃度 Ox-LDL刺激組(0?50?100?150?200?250μg/mL);(3)不同終濃度姜黃素組(0?20?40?80μmol/L);(4)不同終濃度姜黃素組(0?20?40?80μmol/L)+Ox-LDL(100μg/mL)組;(5)Ox-LDL(100μg/mL)+姜黃素組;(6)姜黃素+ZnPPⅨ+Ox-LDL(100μg/mL)組?

1.2.2 反轉(zhuǎn)錄-聚合酶鏈反應(yīng)檢測(cè) HO-1mRNA 按 Trizol試劑盒說(shuō)明提取總RNA?取細(xì)胞RNA 1μL根據(jù)試劑盒說(shuō)明書(shū)逆轉(zhuǎn)錄合成cDNA,進(jìn)行PCR擴(kuò)增?HO-1:上游5′GGGTGACAGAAGAGGCTAAGACC 3′,下 游 5′ AGATTCTCCCCTGCAGAG AGAAG 3′?擴(kuò)增條件:94℃ 變性30s,56℃退火30s,72℃ 延伸45s,循環(huán)30次?電泳條帶采用凝膠成像系統(tǒng)分析?

1.2.3 四甲基偶氮唑鹽(methyl thiazolyl tetrazolium,MTT)法比色測(cè)定VSMC增殖率 VSMC接種96孔培養(yǎng)板中(每孔1×104個(gè)細(xì)胞)孵育24h,換無(wú)血清DMEM培養(yǎng)基24h后棄培養(yǎng)液,按實(shí)驗(yàn)分組加入藥物?20h后加入5mg/mL MTT 10μL,孵育4h,終止反應(yīng)?棄孔內(nèi)培養(yǎng)液上清液加二甲基亞砜100μL,選擇492nm自動(dòng)酶標(biāo)光度計(jì)測(cè)定各孔吸光度值?細(xì)胞增殖率計(jì)算公式:細(xì)胞增殖率(cell viability)= 處理孔A492/對(duì)照孔 A492×100%?

1.2.4 蛋白免疫印跡法(Western blot)檢測(cè) HO-1及增殖細(xì)胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白表達(dá)水平 收集細(xì)胞并加入細(xì)胞裂解液裂解細(xì)胞,離心收集上清液,二喹啉甲酸(bicinchoninic acid,BCA)法測(cè)蛋白濃度?蛋白(30μg)經(jīng)12%十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(sodium dodecyl sulfate polyacrylamide gel electropheresis,SDSPAGE)分離后轉(zhuǎn)移至聚偏(二)氟乙烯(polyvinylidene difluoride,PVDF)膜?5%牛血清清蛋白室溫封閉4h,加入一抗4℃過(guò)夜?二抗室溫孵育2h,最后顯像?

1.3 統(tǒng)計(jì)學(xué)處理 數(shù)據(jù)均采用±s表示,采用單因素方差分析,總體有差異兩兩比較采用最小顯著差數(shù)法?采用SPSS13.0軟件進(jìn)行分析,P0.05為差異有統(tǒng)計(jì)學(xué)意義?

2 結(jié) 果

2.1 大鼠血管平滑肌細(xì)胞鑒定 培養(yǎng)的第3代細(xì)胞經(jīng)免疫細(xì)胞化學(xué)檢測(cè),特異性的α-actin在免疫細(xì)胞化學(xué)染色后,胞漿著色,呈陽(yáng)性反應(yīng),在鏡下可見(jiàn)胞漿內(nèi)大量棕色?與細(xì)胞長(zhǎng)軸平行的纖維細(xì)絲,即平滑肌α肌動(dòng)蛋白,見(jiàn)封3圖1?

2.2 Ox-LDL對(duì)誘導(dǎo)大鼠血管平滑肌細(xì)胞(VSMC)增殖的影響 Ox-LDL呈濃度依賴性(0～150μg/mL)地誘導(dǎo) VSMC增殖,但是當(dāng)達(dá)到200μg/mL刺激組時(shí),細(xì)胞數(shù)目減少,提示到達(dá)此濃度可能具備細(xì)胞毒性?各個(gè)濃度刺激組與空白對(duì)照組相比,細(xì)胞增殖顯著,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),見(jiàn)圖2?

2.3 姜黃素對(duì)Ox-LDL刺激的VSMCs增殖的影響 不同終濃度的姜黃素(40?80μmol/L)可明顯抑制 Ox-LDL(100μg/mL)誘導(dǎo)的細(xì)胞增殖效應(yīng),且呈濃度依賴性;姜黃素(40?80 μmol/L)+Ox-LDL組與 Ox-LDL單獨(dú)作用組相比,細(xì)胞增殖率顯著降低,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),見(jiàn)圖3?

圖2 Ox-LDL對(duì)誘導(dǎo)大鼠血管平滑肌細(xì)胞(VSMC)增殖的影響

2.4 HO-1抑制劑ZnPPⅨ對(duì)姜黃素抑制Ox-LDL所誘導(dǎo)的VSMC增殖的影響 給予HO-1抑制劑ZnPPⅨ預(yù)處理細(xì)胞后,姜黃素抑制Ox-LDL所誘導(dǎo)的VSMCs增殖效應(yīng)減弱;姜黃素+ZnPPⅨ+Ox-LDL組與姜黃素+Ox-LDL組相比,細(xì)胞增殖率增加明顯(P0.05),見(jiàn)圖4?

2.5 姜黃素對(duì)VSMC的HO-1表達(dá)的影響 不同濃度姜黃素組干預(yù)VSMC的HO-1mRNA和蛋白表達(dá)水平明顯高于空白對(duì)照組,實(shí)驗(yàn)重復(fù)3次,見(jiàn)封4圖5?

2.6 姜黃素對(duì)Ox-LDL刺激VSMC的PCNA表達(dá)的影響

相比較于空白對(duì)照組,Ox-LDL可明顯增強(qiáng)PCNA的表達(dá);而姜黃素+Ox-LDL組與Ox-LDL組單獨(dú)作用比較,可明顯抑制PCNA的表達(dá);在加入HO-1抑制劑ZnPPⅨ后,姜黃素+Ox-LDL組的抑制PCNA表達(dá)效應(yīng)可被減弱,實(shí)驗(yàn)重復(fù)3次,見(jiàn)封4圖6?

圖3 姜黃素對(duì) Ox-LDL(100μg/mL)所誘導(dǎo)的VSMC增殖的影響

圖4 ZnPPⅨ對(duì)姜黃素抑制Ox-LDL所誘導(dǎo)的VSMCs增殖的影響

3 討 論

平滑肌細(xì)胞增殖是AS斑塊形成過(guò)程中的重要環(huán)節(jié),大量研究表明動(dòng)脈血管壁增厚至斑塊形成是AS病變的主要病理特征?Ox-LDL促進(jìn)AS形成的作用已被大量的研究所證實(shí)?目前認(rèn)為AS形成初期步驟是LDL進(jìn)入內(nèi)皮損傷部位的血管壁,氧化?誘導(dǎo)多種趨化因子,并激活單核細(xì)胞分泌不同的生長(zhǎng)因子和細(xì)胞因子,刺激平滑肌細(xì)胞遷移?增殖?因此,Ox-LDL在AS中起到關(guān)鍵作用?

HO-1是血紅素降解的起始酶和限速酶,HO-1通過(guò)降解血紅素產(chǎn)生一氧化碳(carbon monoxide,CO)?膽綠素和鐵離子?膽綠素進(jìn)一步經(jīng)膽綠素還原酶作用生成膽紅素,而鐵離子能誘導(dǎo)鐵蛋白的合成[4]?HO-1的這些終產(chǎn)物具有抗炎?抗氧化?抗凋亡和抗血栓的作用;另外,CO和膽色素通過(guò)影響血管平滑肌細(xì)胞?內(nèi)皮細(xì)胞?內(nèi)皮祖細(xì)胞和粒細(xì)胞的增殖?遷移和黏附保護(hù)受損動(dòng)脈血管內(nèi)平衡[5]?姜黃素是中藥姜黃的主要成分,其抗炎?抗氧化?抗腫瘤?抗血栓以及心血管的保護(hù)作用已經(jīng)被證實(shí)[6]?目前已經(jīng)有研究表明姜黃素具有抗細(xì)胞增殖的作用,但是具體機(jī)制還不甚清楚[7-8]?根據(jù)其眾多藥理作用與HO-1保護(hù)作用相似,姜黃素的相關(guān)保護(hù)作用是否經(jīng)過(guò)HO-1介導(dǎo)值得研究?

本研究結(jié)果顯示,Ox-LDL在一定濃度范圍內(nèi)濃度依賴性地誘導(dǎo)血管平滑肌細(xì)胞增殖,當(dāng)超過(guò)一定濃度,具備細(xì)胞毒性?此實(shí)驗(yàn)發(fā)現(xiàn)與Chang等[9]的研究結(jié)果相類似,說(shuō)明了Ox-LDL對(duì)血管平滑肌細(xì)胞增殖影響的特點(diǎn),其機(jī)制可能是通過(guò)激活Ras/Raf/MEK/MAPK通路促進(jìn)細(xì)胞有絲分裂[10]?與之類似,Schwer等[11]也發(fā)現(xiàn)姜黃素 HO-1介導(dǎo)抑制胰腺星狀細(xì)胞的增殖由阻遏ERK1/2磷酸化的機(jī)制實(shí)現(xiàn)?同時(shí),本組也發(fā)現(xiàn)姜黃素可有效抑制Ox-LDL誘導(dǎo)血管平滑肌細(xì)胞增殖,并且此抑制效應(yīng)可被HO-1的抑制劑ZnPPⅨ部分阻斷?這提示HO-1很可能參與了姜黃素抑制Ox-LDL誘導(dǎo)血管平滑肌細(xì)胞增殖的保護(hù)效應(yīng)?同樣,有學(xué)者發(fā)現(xiàn)在球囊損傷的大鼠模型中,HO-1介導(dǎo)NO抑制大鼠血管平滑肌增殖,從而顯著地抑制了內(nèi)膜增生[12]?為了進(jìn)一步證實(shí)此發(fā)現(xiàn),本組觀察了姜黃素對(duì)血管平滑肌細(xì)胞HO-1mRNA和蛋白表達(dá)的影響,發(fā)現(xiàn)姜黃素可顯著地上調(diào)血管平滑肌細(xì)胞HO-1mRNA和蛋白表達(dá)?綜上所得出的實(shí)驗(yàn)結(jié)果,提示姜黃素抗Ox-LDL誘導(dǎo)血管平滑肌細(xì)胞增殖的作用經(jīng)過(guò)HO-1介導(dǎo)?

PCNA又稱周期蛋白,是一種與細(xì)胞增殖周期有關(guān)的核內(nèi)糖蛋白,是DNA復(fù)制的必需成分,其表達(dá)與細(xì)胞增殖活性有關(guān),是使細(xì)胞由靜息期進(jìn)入S期的關(guān)鍵蛋白[13],已成為評(píng)價(jià)細(xì)胞增殖狀態(tài)的一項(xiàng)客觀指標(biāo)?本組研究發(fā)現(xiàn)HO-1抑制劑上調(diào)姜黃素抑制Ox-LDL誘導(dǎo)VSMCs增殖細(xì)胞核抗原高表達(dá),提示HO-1介導(dǎo)的抑制增殖的效應(yīng)可能部分通過(guò)抑制PCNA表達(dá)實(shí)現(xiàn)?另外研究發(fā)現(xiàn)HO-1的抗增殖效應(yīng)通過(guò)上調(diào)細(xì)胞周期蛋白依賴激酶抑制因子P21[14],P21是細(xì)胞周期素蛋白cdk-2抑制蛋白,cdk-2亦是細(xì)胞周期G1和S期調(diào)節(jié)關(guān)鍵?Liu等[15]以含HO-1基因的重組腺病毒轉(zhuǎn)染大鼠主動(dòng)脈平滑肌細(xì)胞,被轉(zhuǎn)染的大鼠主動(dòng)脈平滑肌細(xì)胞以劑量依賴方式表達(dá)HO-1mRNA?HO-1蛋白和提高酶的活性,刺激促凋亡基因p53表達(dá),促進(jìn)VSMCs凋亡而抑制血清刺激的VSMCs增殖?

綜上所述,姜黃素顯著地抑制Ox-LDL誘導(dǎo)的大鼠VSMC增殖,其機(jī)制是由HO-1介導(dǎo)的PCNA表達(dá)下調(diào)實(shí)現(xiàn)的?這為進(jìn)一步研究姜黃素防治AS提供了一定的理論依據(jù)?

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Role of heme oxygenase-1 on curcumin inhibiting rat vascular smooth muscle cells proliferation induced by oxidized low density lipoprotein

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ObjectiveTo investigate the inhibitory effect of curcumin on oxidized(Ox)-low density lipoprotein(LDL)-induced rat vascular smooth muscle cells proliferation and to explore the role of heme oxygenase-1in this process.MethodsThe cultured aortic smooth muscle cells isolated from male Sprague-Dawley rats were verified by immunohistochemistry.The cell viability was assayed by MTT,and reverse transcription-polymerase chain reaction(RT-PCR)was performed to determine the level of HO-1mRNA.In addition,HO-1and PCNA expression were analyzed by Western blotting.ResultsThe Ox-LDL remarkably stimulated VSMCs proliferation in a concentration-dependent manner(0-150μg/ml),however,the doses that exceeded 200mg/ml had a cytotoxic effect.Curcumin also attenuated the VSMCs proliferation effect of Ox-LDL in a concentration-dependent manner(40,80μM),the inhibitory effect was blocked by a HO-1inhibitor(zine protoporphyrinⅨ,ZnPPⅨ).Curcumin significantly upregulated HO-1mRNA and protein expression in a concentration-dependent manner.Furthermore,ZnPPⅨblocked the inhibitory effect of curcumin on Ox-LDL-induced PCNA expression.ConclusionCurcumin inhibits Ox-LDL-induced rat vascular smooth muscle cells proliferation via HO-1-mediated down-regulation of PCNA expression.

curcumin;oxygenases;lipoproteins,LDL

10.3969/j.issn.1671-8348.2012.16.018

A

1671-8348(2012)16-1609-03

△ 通訊作者,Tel:(020)61641508;E-mail:guozhigang126@126.com?

2011-09-11

2011-12-22)

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