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染料木素保护血管紧张素Ⅱ致高血压小鼠心脏纤维化

2016-04-25华晓芳沈艳芳

河北医药 2016年6期
关键词:纤维化心脏小鼠

华晓芳 沈艳芳

445000 湖北省恩施土家族苗族自治州中心医院内科心血管中心(华晓芳),健康体检中心(沈艳芳)



·论著·

染料木素保护血管紧张素Ⅱ致高血压小鼠心脏纤维化

华晓芳沈艳芳

445000湖北省恩施土家族苗族自治州中心医院内科心血管中心(华晓芳),健康体检中心(沈艳芳)

【摘要】目的探讨染料木素在血管紧张素Ⅱ(Ang Ⅱ)诱导的高血压小鼠心脏纤维化中的作用。方法30只雄性C57BL/6J小鼠随机分为对照组、Ang Ⅱ组、Ang Ⅱ+染料木素组,每组10例。Ang Ⅱ组以1 500 ng·kg(-1)·min(-1)连续泵入4.32 μg/μl Ang Ⅱ 7 d,Ang Ⅱ+染料木素组在Ang Ⅱ泵入前5 d开始皮下注射染料木素0.1 μmol/kg,持续至Ang Ⅱ泵入结束,对照组以乙酸代替Ang Ⅱ泵入。心脏超声检测3组小鼠心功能的改变,病理染色检测心脏纤维化程度,实时定量PCR检测collagen Ⅰα、collagen Ⅲ、MMP-9、TGF-β1的mRNA表达量的变化,Western Blot检测3组小鼠心脏自噬的改变。结果染料木素可明显改善心功能,减轻Ang Ⅱ诱导的心脏纤维化程度,Ang Ⅱ+染料木素组LVEF和LVFS与Ang Ⅱ组相比增加,且舒张期E峰运动速率升高(P<0.05),实时定量PCR检测显示Ang Ⅱ+染料木素组collagen Ⅰα、collagen Ⅲ、MMP-9、TGF-β1的mRNA表达量较Ang Ⅱ组显著降低(P<0.05),Western-blot检测结果显示,Ang Ⅱ处理能增加心脏自噬标志物LC3的蛋白表达(P<0.05),而染料木素可显著减轻ISO导致的LC3 Ⅱ表达量的增加(P<0.05)。结论染料木素能减轻Ang Ⅱ诱导的心脏纤维化,其主要是通过降低Ang Ⅱ诱导的心脏自噬而发挥作用的。

【关键词】染料木素;血管紧张素Ⅱ;高血压;小鼠;纤维化,心脏

心肌纤维化是多种常见心血管疾病发展到终末阶段的必然过程,是心肌结构重构的主要表现,其病理变化主要为心肌组织胶原纤维过量沉积、胶原浓度显著升高或胶原成分发生改变[1]。在心肌纤维化过程中,成纤维细胞聚集增多,细胞外基质蛋白大量沉积,可导致心脏室壁僵硬度增加,影响心脏舒张功能,最终发展成为心力衰竭。过度沉积的细胞外基质可损害心肌细胞的机械-电耦合,导致心律失常的发生[2]。研究表明,心肌纤维化与血管紧张素Ⅱ(Ang Ⅱ)、转化生长因子β(TGF-β)、结缔组织生长因子(CTGF)、内皮素-1(ET-1)、血小板源性生长因子(PDGF)等活性物质密切相关[3,4]。上述活性物质均可刺激成纤维细胞分化为肌成纤维细胞,而促进细胞外基质沉积,加剧心肌纤维化发展。目前尚无有效逆转或治疗心肌纤维化的药物,研究抗纤维化药物对延缓各种心血管疾病的发生发展至关重要。本实验用Ang Ⅱ诱导心肌纤维化模型探讨染料木素在心脏纤维化中的作用。

1材料与方法

1.1动物及材料雄性C57BL/6J小鼠30只购买于维通利华实验动物中心(北京),SPF级,体重23~25 g,8~10周龄,动物许可证号:SYXK(京)2011-0028。染料木素购于陕西华昌生物工程有限公司,花白色结晶,纯度98%;血管紧张素Ⅱ购于Sigma,5 mg。植入式胶囊渗透泵购于ALZET,1007D。

1.2实验分组将30只C57BL/6J小鼠编号,随机分为3组,每组10只。对照组:以1 500 ng·kg-1·min-1连续泵入0.01%乙酸7 d;Ang Ⅱ组:以1 500 ng·kg-1·min-1连续泵入4.32 μg/μl Ang Ⅱ 7 d;Ang Ⅱ+染料木素组:在Ang Ⅱ泵入前5 d开始皮下注射染料木素0.1 μmol/kg,持续至Ang Ⅱ 泵入结束。

1.3高血压心肌纤维化模型制作小鼠适应性喂养3 d后用小动物无创血压计(Softron BP98A)测鼠尾血压,高频率超声系统(Vevo 2100)检测左心室射血分数(LVEF)、左心室短轴缩短率(LVFS)以及二尖瓣环运动速度(E/A)各参数后行小鼠背部皮下植入微量泵手术。具体方法:20 mg/kg戊巴比妥钠腹腔注射麻醉小鼠,俯卧于手术台上,背部皮肤除毛,75%乙醇棉球消毒。用剪刀剪开皮肤,钝性分离皮下,植入已预装的Ang Ⅱ或乙酸微量泵,然后缝合皮下、皮肤。

1.4小鼠超声检查采用Vevo 2100超高分辨率小动物超声影像系统(VisualSonics公司),检测LVEF、LVFS以及E’等相关参数评价各组小鼠心脏结构及其功能。

1.5组织病理染色小鼠颈椎脱臼处死后,取左心室中部心肌组织冠状切面,4%多聚甲醛固定,石蜡包埋后制作组织切片(3 mm),采用苦味酸-天狼星红染色(上海榕柏生物有限公司)进行心脏胶原纤维染色。染色完成后于400倍正置显微镜下观察拍照,每个样本随机选取10个区域进行胶原面积的计算,以Image-Pro Plus 6.0图像分析软件计算胶原纤维的含量。

1.6Western-blot检测取3组小鼠心脏组织100 mg,液氮低温研磨,加裂解液100 μl冰浴作用30 min,4℃、12 000 r/min离心15 min。提取总蛋白后,用BCA蛋白定量(上海荔达生物科技有限公司)法定量各组蛋白浓度。以β-actin水平为等量蛋白质上样参照,取20 μg蛋白质样品进行10% SDS-PAGE电泳,并转至硝酸纤维素膜(北京拜尔迪生物技术有限公司)上;以5%脱脂奶粉室温封闭1 h后,TBST缓冲液漂洗3次,加入一抗LC3A/B(1∶1 000,CST,货号:12741)4℃摇床过夜;TBST漂洗3次后加入山羊抗兔IgG二抗(1∶2 000)室温摇床孵育60 min,洗膜。Odyssey双色红外激光成像系统扫描蛋白印迹,检测每组上述蛋白的表达水平。

1.7实时定量PCR检测小鼠心脏组织匀浆后,用Trizol(Life Tech)提取心肌的总RNA,用逆转录试剂盒(Takara)将总RNA逆转录为cDNA。利用ABI 7 500荧光定量PCR仪进行Real time RT-PCR反应,以β-actin为参照比较各组collagen Ⅰα、collagen Ⅲ、MMP-9、TGF-β1基因表达量。见表1。

表1 PCR引物信息

2结果

2.1染料木素可减轻Ang Ⅱ引起的心功能不全小鼠分组给药处理后,行超声检查评价心脏收缩和舒张功能。与对照组相比,Ang Ⅱ处理组LVEF和LVFS减少,舒张期E峰运动速率显著降低(P<0.05)。染料木素治疗组LVEF和LVFS与Ang Ⅱ组相比增加,且舒张期E峰运动速率升高(P<0.05)。见表2。

表1 3组小鼠心脏收缩功能和舒张功能比较 ±s

注:与对照组比较,*P<0.05;与AngⅡ组比较,#P<0.05

2.2染料木素可减轻Ang Ⅱ致高血压小鼠心脏纤维化小鼠心脏组织切片后行苦味酸-天狼星红染色,结果显示Ang Ⅱ处理组与对照组相比心脏胶原纤维显著聚集(P<0.05),而染料木素可减轻Ang Ⅱ导致的胶原纤维沉积(P<0.05)。实时定量PCR检测3组小鼠Collagen Ⅰα、Collagen Ⅲ、MMP-9和TGF β的mRNA表达量,结果显示Ang Ⅱ处理可增加心脏Collagen Ⅰα、Collagen Ⅲ、MMP-9和TGF β mRNA的表达,而染料木素可抑制Collagen Ⅰα、Collagen Ⅲ、MMP-9和TGF β mRNA的表达(P<0.05)。见图1~2。

对照组AngⅡ组AngⅡ+Genistein组

图13组小鼠心脏纤维化水平(苦味酸-天狼星红染色×400)

图2 3组小鼠心脏纤维化水平(实时定量PCR结果)

注:*P<0.05:Ang Ⅱ组vs.对照组;#P<0.05:Ang Ⅱ+染料木素组vs. Ang Ⅱ组

2.3染料木素可减轻Ang Ⅱ引起的心脏自噬Western-blot检测结果显示Ang Ⅱ处理能增加心脏自噬标志物LC3的蛋白表达(P<0.05),而染料木素可显著减轻ISO导致的LC3 Ⅱ表达量的增加(P<0.05)。见图3。

图3 3组小鼠心脏自噬水平(Western-blot检测结果)

3讨论

染料木素又名染料木黄酮、金雀异黄素,是从豆科植物(大豆、葛根、槐等)中提取的一种异黄酮,为含有芳香环的非类固醇化合物,结构与内源性雌激素相似,可与雌激素α、β受体结合,发挥雌激素样作用。研究表明,染料木素具有抗肿瘤[5]、抗氧化[6]、抗骨质疏松[7]、抗动脉粥样硬化[8]、降低心肌兴奋性和自律性、增强收缩力[9]等作用。然而染料木素在心脏纤维化中的作用尚不明确。

纤维化发生于长期的组织修复过程,几乎可累及机体所有系统、器官,是许多慢性疾病的基本病理变化。在组织修复过程中,成纤维细胞活化并迁移到损伤部位,合成、分泌细胞外基质以修复损伤的组织,而成纤维细胞的过度激活会诱发纤维化疾病[10,11]。心肌纤维化与慢性心力衰竭、心律失常等多种心血管疾病的发生密切相关。心肌纤维化过程中伴随着细胞外基质的重塑,胶原蛋白Collagen I、Collagen Ⅲ是细胞外基质的主要成分可出现大量沉积,MMP-9等关键酶是造成Collagen Ⅰ、Collagen Ⅲ增生的主要原因[12]。此外,TGF-β亦可直接诱导细胞外基质蛋白相关基因表达,抑制细胞外基质降解酶的基因表达,促进细胞外基质的进一步沉积。本研究结果表明在Ang Ⅱ诱导的心肌纤维化中Collagen Ⅰα、Collagen Ⅲ、MMP-9和TGF β mRNA的表达,而染料木素处理可抑制Collagen Ⅰα、Collagen Ⅲ、MMP-9和TGF-β mRNA的表达,即染料木素可抑制Ang Ⅱ诱导的心肌纤维化。

自噬是真核生物细胞中特有的进化保守的物质代谢过程,在细胞生长、发育及稳态平衡的维持中发挥关键作用。在自噬过程中,细胞内损伤的蛋白、核酸或细胞器等被双分子层膜结构的自噬小泡包裹,送入溶酶体中进行降解,为细胞修复、再生、重建等提供原料,实现再循环及再利用[13,14]。正常情况下,细胞内自噬水平较低,在缺血缺氧、应激(如创伤、感染)等状态下,细胞启动自噬降解受损生物大分子及细胞器,可起到自我保护的作用,而过度的自噬激活可以导致细胞死亡。严重心脏损伤过程中,心肌细胞的自噬增加,激活细胞内溶酶体酶可导致心肌细胞凋亡增多,成纤维细胞过度激活,加重心功能的恶化[15]。在自噬形成过程中,作为自噬形成标志物的胞浆型LC3(LC3-Ⅰ)会酶解掉一小段多肽,而转变为膜型LC3(LC3-Ⅱ)[16]。本研究证实,Ang Ⅱ刺激可明显增加心脏自噬的形成,而染料木素能够减少自噬体形成,从而减轻Ang Ⅱ对心脏的损伤作用。

本研究表明,染料木素可减轻Ang Ⅱ导致的心脏损伤、维持心脏舒缩功能,同时减少Ang Ⅱ诱导的心脏纤维化。染料木素可能是通过减少心脏自噬来发挥上述作用的。因此染料木素可能成为治疗心脏损伤及心脏纤维化的新药物。

参考文献

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8Schrader C, Ernst IM, Sinnecker H,et al.Genistein as a potential inducer of the anti-atherogenic enzyme paraoxonase-1: studies in cultured hepatocytes in vitro and in rat liver in vivo. J Cell Mol Med,2012,16:2331-2341.

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13Minina EA,Hofius D.Autophagy as initiator or executioner of cell death. Trends Plant Sci,2014,22:S1360-1385.

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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)

【中图分类号】R 972

【文献标识码】A

【文章编号】1002-7386(2016)06-0827-04

通讯作者:沈艳芳,445000湖北省恩施土家族苗族自治州中心医院健康体检中心;

doi:10.3969/j.issn.1002-7386.2016.06.007

E-mail:37629106@qq.com

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