CN116077510A - Tead1抑制剂在制备抗心力衰竭药物中的应用 - Google Patents
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Abstract
本发明公开了TEAD1抑制剂在制备抗心力衰竭药物中的应用。本发明通过特异性敲除成纤细胞TEAD1和TEAD1特异性抑制剂VT103抑制心脏成纤维细胞中的TEAD1活性，抑制TEAD1能够抑制wnt信号通路和成纤维细胞向肌成纤维细胞的转化，减轻心肌纤维化和心肌肥厚，显著改善主动脉弓缩窄(transverse aortic constriction，TAC)诱导的心功能异常；本发明的结果表明，对于临床上慢性心力衰竭的病人给予TEAD1抑制剂能够阻断心脏重构，改善心脏收缩和舒张功能，提高患者的生活质量和远期预后，具有重要的临床意义。
Description
技术领域
本发明涉及TEAD1抑制剂在制备抗心力衰竭药物中的应用，属于生物医药技术领域。
背景技术
心力衰竭(heart failure，HF)是各种心血管疾病的终末阶段，已成为当今世界重大公共卫生问题之一。尽管临床上抑制神经内分泌过度激活等“金三角”药物治疗使心衰患者生存率有所提高，但其生活质量以及远期预后仍然很差。心脏重构已被证实是心力衰竭发生和发展的重要病理生理基础。在大体形态水平，心肌重构多表现为局部或大部分甚至整个心室壁或/和心房壁增厚、质量增加。在细胞水平，不能分裂的单个心肌细胞肥大，间质成分如成纤维细胞增殖，胶原合成和分泌增加，并伴随着心肌细胞凋亡，引起心肌组织成分发生改变。在分子水平，促进心肌纤维化的基因表达增加，如结缔组织生长因子CTGF、转化生长因TGFβ，Ⅰ型和Ⅲ型胶原等，使心室壁的僵硬度增加，心肌顺应性下降。尽管心脏重构最初是一种代偿机制，长期持续性的心脏重构能够明显增加心力衰竭的发病率和病死率。目前，对于心脏重构导致的心力衰竭临床上尚无十分有效的防治方法。因此，发现阻断心脏重构导致心力衰竭的特异性分子以及分子通路，寻找防治心力衰竭的药物靶点具有非常重要的理论和临床意义。
TEAD1(TEA domain Family memher 1)是一个具有TEA结构域的转录因子，可结合某些肌肉特异表达基因启动子区域的M-CAT元件，正调控或负调控基因的表达。TEAD1在除血液细胞外所有组织细胞中丰富表达，能够调控对于胚胎发育、心脏发生、肌细胞生成、血管损伤及细胞分化等至关重要的基因表达。但TEAD1在心脏重构中的作用目前尚未见相关报道。
发明内容
本发明的目的是：针对目前心脏重构导致的心力衰竭临床上尚无十分有效的防治方法，本发明旨在通过靶向抑制TEAD1改善心脏重构导致的心力衰竭，结果表明，通过给予TEAD1抑制剂能够减轻心肌纤维化和心肌肥厚，显著改善心功能异常。
为了实现上述目的，本发明提供了抑制TEAD1表达或活性的试剂在制备抗心力衰竭药物中的应用。所述抗心力衰竭指的是心脏重构导致的心力衰竭。
优选地，所述抑制TEAD1表达或活性的试剂包括TEAD1抑制剂。
优选地，所述TEAD1抑制剂为VT103，其化学结构式如下：
长期的高血压、心肌缺血等刺激会诱发心力衰竭，但这一过程往往会伴随着心脏重构的发生。心肌纤维化和心肌肥厚等病理性重构严重影响心脏的收缩和舒张功能，加速心力衰竭的进展。因此，本发明着眼于靶向抑制TEAD1这一转录因子，抑制wnt信号通路和成纤维细胞向肌成纤维细胞的转化，阻断心脏重构的关键环节，改善心脏功能。
与现有技术相比，本发明的有益效果在于：
本发明通过特异性敲除成纤细胞TEAD1和TEAD1特异性抑制剂VT103抑制心脏成纤维细胞中的TEAD1活性，抑制TEAD1能够抑制wnt信号通路和成纤维细胞向肌成纤维细胞的转化，减轻心肌纤维化和心肌肥厚，显著改善主动脉弓缩窄(transverse aorticconstriction，TAC)诱导的心功能异常；本发明的结果表明，对于临床上慢性心力衰竭的病人给予TEAD1抑制剂能够阻断心脏重构，改善心脏收缩和舒张功能，提高患者的生活质量和远期预后，具有重要的临床意义。
附图说明
图1为动物体内给予TEAD1抑制剂VT103处理实验方案的流程图；
图2展示了心脏成纤维细胞中特异性敲除TEAD1后可以改善TAC引起的心肌重构和心功能恶化的结果；A：动物模型构建流程；B：M型超声结果显示，成纤维细胞中TEAD1特异性敲除后可以改善TAC引起的心功能恶化；C：成纤维细胞中TEAD1特异性敲除后可以提高心衰小鼠的射血分数(EF％)和缩短分数(FS％)；D：成纤维细胞中TEAD1特异性敲除后可以降低心重/体重(HW/BW)和心重/胫骨长度(HW/TL)；E：各组小鼠生存曲线；F：天狼猩红显示各组小鼠纤维化程度；G：WGA显示各组小鼠心肌细胞肥大程度；H：Western blot检测不同处理组小鼠细胞外基质蛋白的表达情况；I：免疫荧光双标(红色-collagen I，绿色-collagenIII)检测不同处理组小鼠的纤维化程度，50μM；J：心衰相关指标(ANP/BNP/β-MHC)的mRNA表达水平；
图3展示了TEAD1抑制剂VT103可以改善TAC引起的心肌重构和心功能恶化的结果；A：动物模型构建流程；B：M型超声结果显示，VT103可以改善TAC引起的心功能恶化；C：VT103可以提高心衰小鼠的射血分数(EF％)和缩短分数(FS％)；D：VT103可以降低心重/体重(HW/BW)和心重/胫骨长度(HW/TL)；E：各组小鼠生存曲线；F：HE染色显示各组小鼠心脏肥厚和扩张程度；G：天狼猩红显示各组小鼠纤维化程度；H：WGA显示各组小鼠心肌细胞肥大程度；I：Western blot检测不同处理组小鼠细胞外基质蛋白的表达情况；J：心衰相关指标(ANP/BNP/β-MHC)的mRNA表达水平。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
以下实施例中涉及的小鼠购自赛业生物科技公司，TEAD1特异性抑制剂VT103购自MCE(MedChemExpress)公司，动物实验已通过复旦大学附属中山医院伦理委员会批准。
TEAD1特异性抑制剂VT103的化学结构式如下：
实施例
本实施例提供了TEAD1抑制剂在抗心力衰竭中的应用：
(一)实验方法：
1、主动脉弓缩窄模型(TAC模型)的构建
使用雄性C57BL/6野生型小鼠(6-8周大，WT型)。腹腔注射1％戊巴比妥钠0.15ml麻醉小鼠。用眼科剪在小鼠颈部和胸部中线位置纵向切开皮肤0.5～1cm，使用眼科镊轻轻分离结缔组织和气管中线两侧肌肉。用眼科剪从锁骨处开始切至第二肋骨(约5mm)。使用显微镊将胸腺两叶彼此分离，此时可清晰看到主动脉弓和两条颈动脉。用显微镊从左颈总动脉和右无名动脉之间，主动脉弓下方轻轻穿过，夹住6-0尼龙缝合线小心拉出。将自制L型26G垫针防至主动脉弓旁，将主动脉弓与L型垫针结扎固定，打三个结确保结扎稳固，小心抽去垫针。主动脉弓狭窄成功后可观察到右颈总动脉搏动显著增强。简单间断缝合肌肉和皮肤，碘伏消毒伤口后置于保温台上恢复，苏醒后放回笼具。假手术组方法一致，只穿线不结扎。
2、小鼠心脏超声
小鼠脱毛后，用异氟醚麻醉后固定在加热垫上(1％-2％)。小心控制异氟醚流量，使心率维持在450次/分左右。采用配备30mHz线性换能器的二维(2-D)引导M型超声心动图(VisualSonics Vevo 2100，加拿大)评估心脏的几何结构和功能。获得胸骨旁长轴视图，同时记录心率。得到心脏几何形状和功能指标：EF、缩短分数(FS)。每个测量指标至少经过5个连续的心脏周期取平均值。
3、天狼猩红染色
心肌组织分离，福尔马林固定，并石蜡包埋，切片厚度为6-7μm。天狼星红染色液滴染1h，流水稍微冲洗，去除切片表面染液，之后苏木素染色液染核8～10min，流水冲洗10min。常规脱水透明，中性树胶封固。在光学显微镜下观察，胶原纤维呈红色，细胞核蓝色，其他成分呈黄色。并计算胶原体积分数(CVF)，计算公式：CVF(％)＝平均胶原面积/总区面积×100％。
3、心脏成纤维细胞中特异性敲除TEAD1实验
(1)构建TEAD1-floxed(TEAD1fl/fl)小鼠，与Jackson实验室引进的含心脏成纤维细胞相对特异启动子(Col1a2)的Cre小鼠Col1a2-CreERT2杂交，得到TEAD1fl/fl Col1a2小鼠，该小鼠腹腔注射他莫昔芬(TAM)后能够在成纤维细胞中特异性敲除TEAD1。
(2)将TEAD1fl/fl Col1a2和TEAD1fl/fl同窝对照小组腹腔注射20mg/kg的TAM，每天注射一次，连续给药5天。注射完毕后随机分组，TAC组构建主动脉弓缩窄模型，sham组只穿线不结扎。共得到4组：TEAD1fl/fl+sham；TEAD1fl/fl+TAC；TEAD1fl/fl Col1a2+sham；TEAD1fl/flCol1a2+TAC。
(3)TAC手术后28天小鼠心脏超声检测心功能，HE染色、天狼猩红染色以及WGA染色检测心肌纤维化和心肌细胞肥大水平，Western blot检测细胞外基质蛋白(Fibronectin/TGF-β1/α-SMA/Galectin-3)的表达情况，qPCR检测心衰相关指标(ANP/BNP/β-MHC)的mRNA表达水平。
4、TEAD1抑制剂VT103实验(方案流程如图1所示)
(1)C57BL/6小鼠随机分组，TAC组构建主动脉弓缩窄模型，sham组只穿线不结扎。
(2)手术后3天随机分组，处理组给予30mg/ml的VT103，采用腹腔注射，每次注射0.1ml，每天一次。对照组注射0.1ml PBS，注射方法和频率与实验组保持一致。连续给药28天。
(3)给药结束后小鼠心脏超声检测心功能，HE染色、天狼猩红染色以及WGA染色检测心肌纤维化和心肌细胞肥大水平，Western blot检测细胞外基质蛋白(Fibronectin/TGF-β1/α-SMA/Galectin-3)的表达情况，qPCR检测心衰相关指标(ANP/BNP/β-MHC)的mRNA表达水平。
(二)实验结果
1、心脏成纤维细胞中特异性敲除TEAD1实验结果
(1)主动脉缩窄(TAC)诱导心衰模型的建立及给药方式如图2A所示。不同实验组小鼠的心功能评估以小鼠左室心肌射血分数和(EF％)和左心窒的短轴缩短率(FS％)为标准。如图2B和2C所示，结果显示，成纤维细胞中TEAD1特异性敲除后可以改善TAC引起的心功能恶化，提高心衰小鼠的射血分数(EF％)和缩短分数(FS％)。
(2)心肌纤维化和心肌细胞肥大是心脏重构的重要特征。实验中使用了天狼猩红染色以及WGA染色评估不同处理组的心肌纤维化肥大水平。结果显示，与对照组相比，成纤维细胞中TEAD1特异性敲除后能够显著减轻TAC引起的心肌纤维化和心肌细胞肥大(图2F-G)。
(3)Fibronectin、TGF-β1、α-SMA、Galectin-3等细胞外基质蛋白是评估心肌纤维化的重要指标，ANP、BNP和β-MHC水平是评估心力衰竭程度的重要指标。采用Western blot检测了Fibronectin、TGF-β1、α-SMA、Galectin-3的表达情况，采用免疫荧光双标(红色-collagen I，绿色-collagen III)检测不同处理组小鼠的纤维化程度，qPCR检测了小鼠心脏组织中ANP、BNP和β-MHC表达量。结果显示，与对照组相比，成纤维细胞中TEAD1特异性敲除后能够显著降低Fibronectin、TGF-β1、α-SMA、Galectin-3、ANP、BNP和β-MHC的表达量并能减轻心肌纤维化(图2H-J)。
2、TEAD1抑制剂VT103实验结果
(1)主动脉缩窄(TAC)诱导心衰模型的建立及给药方式如图3A所示。不同干预组小鼠的心功能评估以小鼠左室心肌射血分数和(EF％)和左心窒的短轴缩短率(FS％)为标准。如图3B和3C所示，结果显示，与对照组sham相比，TAC组小鼠的心肌EF值和FS值显著降低，TEAD1特异性抑制剂VT103能够显著增加TAC后的EF和FS值。表明心脏成纤维细胞中特异性敲除TEAD1后可以改善TAC引起的心肌重构和心功能恶化。
(2)心肌纤维化和心肌细胞肥大是心脏重构的重要特征。实验中使用了HE染色、天狼猩红染色以及WGA染色评估不同处理组的心肌纤维化肥大水平。结果显示，与对照组相比，TEAD1特异性抑制剂VT103能够显著减轻TAC引起的心肌纤维化和心肌细胞肥大(图3G-H)。
(3)Fibronectin、TGF-β1、α-SMA、Galectin-3等细胞外基质蛋白是评估心肌纤维化的重要指标，ANP、BNP和β-MHC水平是评估心力衰竭程度的重要指标。采用Western blot检测了Fibronectin、TGF-β1、α-SMA、Galectin-3的表达情况，qPCR检测了小鼠心脏组织中ANP、BNP和β-MHC表达量。研究结果显示，与对照组相比，TEAD1特异性抑制剂VT103能够显著降低Fibronectin、TGF-β1、α-SMA、Galectin-3、ANP、BNP和β-MHC的表达量(图3I-J)。
上述实施例仅为本发明的优选实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (3)
1.抑制TEAD1表达或活性的试剂在制备抗心力衰竭药物中的应用。所述抗心力衰竭指的是心脏重构导致的心力衰竭。
2.如权利要求1所述的应用，其特征在于，所述抑制TEAD1表达或活性的试剂包括TEAD1抑制剂。
3.如权利要求2所述的应用，其特征在于，所述TEAD1抑制剂为VT103，其化学结构式如下：
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