華曉芳　沈艷芳

445000　湖北省恩施土家族苗族自治州中心醫(yī)院內(nèi)科心血管中心(華曉芳)，健康體檢中心(沈艷芳)

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·論著·

染料木素保護(hù)血管緊張素Ⅱ致高血壓小鼠心臟纖維化

華曉芳沈艷芳

445000湖北省恩施土家族苗族自治州中心醫(yī)院內(nèi)科心血管中心(華曉芳)，健康體檢中心(沈艷芳)

【摘要】目的探討染料木素在血管緊張素Ⅱ(Ang Ⅱ)誘導(dǎo)的高血壓小鼠心臟纖維化中的作用。方法30只雄性C57BL/6J小鼠隨機(jī)分為對照組、Ang Ⅱ組、Ang Ⅱ+染料木素組，每組10例。Ang Ⅱ組以1 500 ng·kg(-1)·min(-1)連續(xù)泵入4.32 μg/μl Ang Ⅱ 7 d，Ang Ⅱ+染料木素組在Ang Ⅱ泵入前5 d開始皮下注射染料木素0.1 μmol/kg，持續(xù)至Ang Ⅱ泵入結(jié)束，對照組以乙酸代替Ang Ⅱ泵入。心臟超聲檢測3組小鼠心功能的改變，病理染色檢測心臟纖維化程度，實(shí)時(shí)定量PCR檢測collagen Ⅰα、collagen Ⅲ、MMP-9、TGF-β1的mRNA表達(dá)量的變化，Western Blot檢測3組小鼠心臟自噬的改變。結(jié)果染料木素可明顯改善心功能，減輕Ang Ⅱ誘導(dǎo)的心臟纖維化程度，Ang Ⅱ+染料木素組LVEF和LVFS與Ang Ⅱ組相比增加，且舒張期E峰運(yùn)動速率升高(P<0.05)，實(shí)時(shí)定量PCR檢測顯示Ang Ⅱ+染料木素組collagen Ⅰα、collagen Ⅲ、MMP-9、TGF-β1的mRNA表達(dá)量較Ang Ⅱ組顯著降低(P<0.05)，Western-blot檢測結(jié)果顯示，Ang Ⅱ處理能增加心臟自噬標(biāo)志物L(fēng)C3的蛋白表達(dá)(P<0.05)，而染料木素可顯著減輕ISO導(dǎo)致的LC3 Ⅱ表達(dá)量的增加(P<0.05)。結(jié)論染料木素能減輕Ang Ⅱ誘導(dǎo)的心臟纖維化，其主要是通過降低Ang Ⅱ誘導(dǎo)的心臟自噬而發(fā)揮作用的。

【關(guān)鍵詞】染料木素；血管緊張素Ⅱ；高血壓；小鼠；纖維化，心臟

心肌纖維化是多種常見心血管疾病發(fā)展到終末階段的必然過程，是心肌結(jié)構(gòu)重構(gòu)的主要表現(xiàn)，其病理變化主要為心肌組織膠原纖維過量沉積、膠原濃度顯著升高或膠原成分發(fā)生改變[1]。在心肌纖維化過程中，成纖維細(xì)胞聚集增多，細(xì)胞外基質(zhì)蛋白大量沉積，可導(dǎo)致心臟室壁僵硬度增加，影響心臟舒張功能，最終發(fā)展成為心力衰竭。過度沉積的細(xì)胞外基質(zhì)可損害心肌細(xì)胞的機(jī)械-電耦合，導(dǎo)致心律失常的發(fā)生[2]。研究表明，心肌纖維化與血管緊張素Ⅱ(Ang Ⅱ)、轉(zhuǎn)化生長因子β(TGF-β)、結(jié)締組織生長因子(CTGF)、內(nèi)皮素-1(ET-1)、血小板源性生長因子(PDGF)等活性物質(zhì)密切相關(guān)[3,4]。上述活性物質(zhì)均可刺激成纖維細(xì)胞分化為肌成纖維細(xì)胞，而促進(jìn)細(xì)胞外基質(zhì)沉積，加劇心肌纖維化發(fā)展。目前尚無有效逆轉(zhuǎn)或治療心肌纖維化的藥物，研究抗纖維化藥物對延緩各種心血管疾病的發(fā)生發(fā)展至關(guān)重要。本實(shí)驗(yàn)用Ang Ⅱ誘導(dǎo)心肌纖維化模型探討染料木素在心臟纖維化中的作用。

1材料與方法

1.1動物及材料雄性C57BL/6J小鼠30只購買于維通利華實(shí)驗(yàn)動物中心(北京)，SPF級，體重23～25 g，8～10周齡，動物許可證號：SYXK(京)2011-0028。染料木素購于陜西華昌生物工程有限公司，花白色結(jié)晶，純度98%；血管緊張素Ⅱ購于Sigma，5 mg。植入式膠囊滲透泵購于ALZET，1007D。

1.2實(shí)驗(yàn)分組將30只C57BL/6J小鼠編號，隨機(jī)分為3組，每組10只。對照組：以1 500 ng·kg-1·min-1連續(xù)泵入0.01%乙酸7 d；Ang Ⅱ組：以1 500 ng·kg-1·min-1連續(xù)泵入4.32 μg/μl Ang Ⅱ 7 d；Ang Ⅱ+染料木素組：在Ang Ⅱ泵入前5 d開始皮下注射染料木素0.1 μmol/kg，持續(xù)至Ang Ⅱ 泵入結(jié)束。

1.3高血壓心肌纖維化模型制作小鼠適應(yīng)性喂養(yǎng)3 d后用小動物無創(chuàng)血壓計(jì)(Softron BP98A)測鼠尾血壓，高頻率超聲系統(tǒng)(Vevo 2100)檢測左心室射血分?jǐn)?shù)(LVEF)、左心室短軸縮短率(LVFS)以及二尖瓣環(huán)運(yùn)動速度(E/A)各參數(shù)后行小鼠背部皮下植入微量泵手術(shù)。具體方法：20 mg/kg戊巴比妥鈉腹腔注射麻醉小鼠，俯臥于手術(shù)臺上，背部皮膚除毛，75%乙醇棉球消毒。用剪刀剪開皮膚，鈍性分離皮下，植入已預(yù)裝的Ang Ⅱ或乙酸微量泵，然后縫合皮下、皮膚。

1.4小鼠超聲檢查采用Vevo 2100超高分辨率小動物超聲影像系統(tǒng)(VisualSonics公司)，檢測LVEF、LVFS以及E’等相關(guān)參數(shù)評價(jià)各組小鼠心臟結(jié)構(gòu)及其功能。

1.5組織病理染色小鼠頸椎脫臼處死后，取左心室中部心肌組織冠狀切面，4%多聚甲醛固定，石蠟包埋后制作組織切片(3 mm)，采用苦味酸-天狼星紅染色(上海榕柏生物有限公司)進(jìn)行心臟膠原纖維染色。染色完成后于400倍正置顯微鏡下觀察拍照，每個(gè)樣本隨機(jī)選取10個(gè)區(qū)域進(jìn)行膠原面積的計(jì)算，以Image-Pro Plus 6.0圖像分析軟件計(jì)算膠原纖維的含量。

1.6Western-blot檢測取3組小鼠心臟組織100 mg，液氮低溫研磨，加裂解液100 μl冰浴作用30 min，4℃、12 000 r/min離心15 min。提取總蛋白后，用BCA蛋白定量(上海荔達(dá)生物科技有限公司)法定量各組蛋白濃度。以β-actin水平為等量蛋白質(zhì)上樣參照，取20 μg蛋白質(zhì)樣品進(jìn)行10% SDS-PAGE電泳，并轉(zhuǎn)至硝酸纖維素膜(北京拜爾迪生物技術(shù)有限公司)上；以5%脫脂奶粉室溫封閉1 h后，TBST緩沖液漂洗3次，加入一抗LC3A/B(1∶1 000，CST，貨號：12741)4℃搖床過夜；TBST漂洗3次后加入山羊抗兔IgG二抗(1∶2 000)室溫?fù)u床孵育60 min，洗膜。Odyssey雙色紅外激光成像系統(tǒng)掃描蛋白印跡，檢測每組上述蛋白的表達(dá)水平。

1.7實(shí)時(shí)定量PCR檢測小鼠心臟組織勻漿后，用Trizol(Life Tech)提取心肌的總RNA，用逆轉(zhuǎn)錄試劑盒(Takara)將總RNA逆轉(zhuǎn)錄為cDNA。利用ABI 7 500熒光定量PCR儀進(jìn)行Real time RT-PCR反應(yīng)，以β-actin為參照比較各組collagen Ⅰα、collagen Ⅲ、MMP-9、TGF-β1基因表達(dá)量。見表1。

表1　PCR引物信息

2結(jié)果

2.1染料木素可減輕Ang Ⅱ引起的心功能不全小鼠分組給藥處理后，行超聲檢查評價(jià)心臟收縮和舒張功能。與對照組相比，Ang Ⅱ處理組LVEF和LVFS減少，舒張期E峰運(yùn)動速率顯著降低(P<0.05)。染料木素治療組LVEF和LVFS與Ang Ⅱ組相比增加，且舒張期E峰運(yùn)動速率升高(P<0.05)。見表2。

表1　3組小鼠心臟收縮功能和舒張功能比較　±s

注：與對照組比較，*P<0.05；與AngⅡ組比較，#P<0.05

2.2染料木素可減輕Ang Ⅱ致高血壓小鼠心臟纖維化小鼠心臟組織切片后行苦味酸-天狼星紅染色，結(jié)果顯示Ang Ⅱ處理組與對照組相比心臟膠原纖維顯著聚集(P<0.05)，而染料木素可減輕Ang Ⅱ?qū)е碌哪z原纖維沉積(P<0.05)。實(shí)時(shí)定量PCR檢測3組小鼠Collagen Ⅰα、Collagen Ⅲ、MMP-9和TGF β的mRNA表達(dá)量，結(jié)果顯示Ang Ⅱ處理可增加心臟Collagen Ⅰα、Collagen Ⅲ、MMP-9和TGF β mRNA的表達(dá)，而染料木素可抑制Collagen Ⅰα、Collagen Ⅲ、MMP-9和TGF β mRNA的表達(dá)(P<0.05)。見圖1～2。

對照組AngⅡ組AngⅡ+Genistein組

圖13組小鼠心臟纖維化水平(苦味酸-天狼星紅染色×400)

圖2　3組小鼠心臟纖維化水平(實(shí)時(shí)定量PCR結(jié)果)

注:*P<0.05：Ang Ⅱ組vs.對照組；#P<0.05：Ang Ⅱ+染料木素組vs. Ang Ⅱ組

2.3染料木素可減輕Ang Ⅱ引起的心臟自噬Western-blot檢測結(jié)果顯示Ang Ⅱ處理能增加心臟自噬標(biāo)志物L(fēng)C3的蛋白表達(dá)(P<0.05)，而染料木素可顯著減輕ISO導(dǎo)致的LC3 Ⅱ表達(dá)量的增加(P<0.05)。見圖3。

圖3　3組小鼠心臟自噬水平(Western-blot檢測結(jié)果)

3討論

染料木素又名染料木黃酮、金雀異黃素，是從豆科植物(大豆、葛根、槐等)中提取的一種異黃酮，為含有芳香環(huán)的非類固醇化合物，結(jié)構(gòu)與內(nèi)源性雌激素相似，可與雌激素α、β受體結(jié)合，發(fā)揮雌激素樣作用。研究表明，染料木素具有抗腫瘤[5]、抗氧化[6]、抗骨質(zhì)疏松[7]、抗動脈粥樣硬化[8]、降低心肌興奮性和自律性、增強(qiáng)收縮力[9]等作用。然而染料木素在心臟纖維化中的作用尚不明確。

纖維化發(fā)生于長期的組織修復(fù)過程，幾乎可累及機(jī)體所有系統(tǒng)、器官，是許多慢性疾病的基本病理變化。在組織修復(fù)過程中，成纖維細(xì)胞活化并遷移到損傷部位，合成、分泌細(xì)胞外基質(zhì)以修復(fù)損傷的組織，而成纖維細(xì)胞的過度激活會誘發(fā)纖維化疾病[10,11]。心肌纖維化與慢性心力衰竭、心律失常等多種心血管疾病的發(fā)生密切相關(guān)。心肌纖維化過程中伴隨著細(xì)胞外基質(zhì)的重塑，膠原蛋白Collagen I、Collagen Ⅲ是細(xì)胞外基質(zhì)的主要成分可出現(xiàn)大量沉積，MMP-9等關(guān)鍵酶是造成Collagen Ⅰ、Collagen Ⅲ增生的主要原因[12]。此外，TGF-β亦可直接誘導(dǎo)細(xì)胞外基質(zhì)蛋白相關(guān)基因表達(dá)，抑制細(xì)胞外基質(zhì)降解酶的基因表達(dá)，促進(jìn)細(xì)胞外基質(zhì)的進(jìn)一步沉積。本研究結(jié)果表明在Ang Ⅱ誘導(dǎo)的心肌纖維化中Collagen Ⅰα、Collagen Ⅲ、MMP-9和TGF β mRNA的表達(dá)，而染料木素處理可抑制Collagen Ⅰα、Collagen Ⅲ、MMP-9和TGF-β mRNA的表達(dá)，即染料木素可抑制Ang Ⅱ誘導(dǎo)的心肌纖維化。

自噬是真核生物細(xì)胞中特有的進(jìn)化保守的物質(zhì)代謝過程，在細(xì)胞生長、發(fā)育及穩(wěn)態(tài)平衡的維持中發(fā)揮關(guān)鍵作用。在自噬過程中，細(xì)胞內(nèi)損傷的蛋白、核酸或細(xì)胞器等被雙分子層膜結(jié)構(gòu)的自噬小泡包裹，送入溶酶體中進(jìn)行降解，為細(xì)胞修復(fù)、再生、重建等提供原料，實(shí)現(xiàn)再循環(huán)及再利用[13,14]。正常情況下，細(xì)胞內(nèi)自噬水平較低，在缺血缺氧、應(yīng)激(如創(chuàng)傷、感染)等狀態(tài)下，細(xì)胞啟動自噬降解受損生物大分子及細(xì)胞器，可起到自我保護(hù)的作用，而過度的自噬激活可以導(dǎo)致細(xì)胞死亡。嚴(yán)重心臟損傷過程中，心肌細(xì)胞的自噬增加，激活細(xì)胞內(nèi)溶酶體酶可導(dǎo)致心肌細(xì)胞凋亡增多，成纖維細(xì)胞過度激活，加重心功能的惡化[15]。在自噬形成過程中，作為自噬形成標(biāo)志物的胞漿型LC3(LC3-Ⅰ)會酶解掉一小段多肽，而轉(zhuǎn)變?yōu)槟ば蚅C3(LC3-Ⅱ)[16]。本研究證實(shí)，Ang Ⅱ刺激可明顯增加心臟自噬的形成，而染料木素能夠減少自噬體形成，從而減輕Ang Ⅱ?qū)π呐K的損傷作用。

本研究表明，染料木素可減輕Ang Ⅱ?qū)е碌男呐K損傷、維持心臟舒縮功能，同時(shí)減少Ang Ⅱ誘導(dǎo)的心臟纖維化。染料木素可能是通過減少心臟自噬來發(fā)揮上述作用的。因此染料木素可能成為治療心臟損傷及心臟纖維化的新藥物。

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Effects of genistein on cardiac fibrosis of hypertensive mice induced by angiotensin Ⅱ

HUAXiaofang*,SHENYanfang.

*DepartmentofCardiology,CentralHospitalofEnshiTujiaNationalityandMiaoNationalityAutonomousPrefecture,Hubei,Enshi445000,China

【Abstract】ObjectiveTo investigate the effects of genistein on cardiac fibrosis of hypertensive mice induced by angiotensin Ⅱ(Ang Ⅱ).MethodsThirty male C57BL/6J mice were randomly divided into control group, Ang II group and Ang Ⅱ+genistein group,with 10 mice in each group. The animal models with cardiac fibrosis were induced by subcutaneous continuous pumping with Ang Ⅱ 1500ng/kg for 7 days, however, the mice in Ang Ⅱ+genistein group were injected subcutaneously with genistein 0.1μmol/kg on 5 days before pumping Ang Ⅱ, continuously to the end of pumping Ang Ⅱ. The mice in control group were given ethanoic acid instead of pumping Ang Ⅱ. Then the changes of cardiac function of the mice were detected by cardiac echo-cardiography,the severity of cardiac fibrosis was determined by pathological staining, the expression levels of collagen Ⅰα,collagen Ⅲ,MMP-9,TGF-β1mRNA were measured by Real-time PCR,moreover, the changes of cardiac autophagy were detected by Western Blot for three groups.ResultsGenistein could obviously improve the cardiac function of mice and relieve the severity of cardiac fibrosis induced by genistein. As compared with those in Ang Ⅱ group, the LVEF and LVFS in Ang Ⅱ+genistein group were increased,furthermore, E peak motion velocity during diastole was significantly increased (P<0.05). The expression levels of collagen Ⅰα, collagen Ⅲ, MMP-9 and TGF-β1 mRNA in Ang Ⅱ+genistein group were significantly decreased, as compared with those in Ang Ⅱ group (P<0.05). The results by Western Blot showed that Ang Ⅱ could obviously enhance the expression levels of cardiac autophagy marker-LC3 protein (P<0.05), however, genistein could significantly alleviate the increase of expression levels of LC3 Ⅱ caused by ISO (P<0.05).ConclusionGenistein can alleviate cardiac fibrosis induced by Ang Ⅱ,and its action mechanism is mainly by improving the cardiac autophagy induced by Ang Ⅱ.

【Key words】genistein；angiotensin Ⅱ；hypertension；mice；cardiac fibrosis

(收稿日期：2015-07-18)

【中圖分類號】R 972

【文獻(xiàn)標(biāo)識碼】A

【文章編號】1002-7386(2016)06-0827-04

通訊作者：沈艷芳，445000湖北省恩施土家族苗族自治州中心醫(yī)院健康體檢中心；

doi:10.3969/j.issn.1002-7386.2016.06.007

E-mail:37629106@qq.com