CN108245678B - 预防或延缓心脏衰老的方法和药物 - Google Patents
预防或延缓心脏衰老的方法和药物 Download PDFInfo
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- A61K31/47—Quinolines; Isoquinolines
- A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
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Abstract
本发明公开了一种预防或延缓心脏衰老的方法和药物。本发明发现心脏的衰老信号为TGF‑β，它能够启动下游的信号通路，控制表观遗传修饰H4K20me3水平进而调控衰老进程，并证明这条TGF‑β/H4K20me3衰老通路特异地存在于心脏中。TGF‑β信号的细胞膜受体抑制剂可以阻断这条衰老通路，并特异性地在个体水平抑制心脏衰老。从而本发明提出通过抑制TGF‑β衰老信号膜受体可干预心脏衰老过程。
Description
技术领域
本发明涉及生物医药领域，具体涉及一种与心脏衰老相关的信号分子及其抑制剂的应用。
背景技术
人口老龄化是当今世界的重大议题，衰老疾病的发生给社会和人类健康带来了诸多严重挑战。心血管相关疾病是当今世界上每年因此而死亡人数最多的病因。多种多样的心血管疾病的发生和衰老有着极为密切的关系。如心力衰竭是各种心脏疾病致心功能不全的临床综合征，也是大多数心血管疾病的最终归宿。目前全球心力衰竭患者的数量已高达4千多万，并且以每年200万的速度快速递增。由于心衰的发生和和衰老有着极为密切的关系，在老年人群中，患病率高到20％以上，严重影响老年人的生活质量和寿命。因此，对心脏衰老进行化学小分子干预可预防老年心脏病的发生，改善老年人的晚年生活质量，并有助于衰老相关心脏病的治疗。
细胞衰老是被精密调控的程序化过程。在这个过程中，表观遗传学已被证明起着非常重要的作用。个体外部环境因素或体内灯衰老信号可通过信号转导通路诱导基因组表观修饰的改变，进而导致基因表达和基因组稳定性的改变，最终促发衰老的发生。但是，何种衰老信号通过怎样的信号通路来控制衰老中的表观遗传变化目前还不清楚。何种信号特异的调控某种器官衰老也不清楚，而这些问题对于个体内器官的衰老的发生并进行化学小分子干预是至关重要的。
发明内容
本发明旨在阐明衰老信号诱导心脏细胞表观修饰改变并导致心脏衰老的信号及其通路，在此基础上，提供用于预防细胞衰老和心脏老化的方法和药物。
本发明发现心脏的衰老信号为TGF-β，即转化生长因子-β(transforming growthfactor-β)。已知TGF-β通过结合其细胞膜上的受体将信号传入细胞内，导致基因表达的改变从而在多种细胞重大生命活动如增殖和分化中起作用。本发明发现它能够启动下游的信号通路导致心脏细胞中表观遗传的改变从而促发衰老。TGF-β信号的细胞膜受体抑制剂可以阻断这条衰老通路，并特异性地在个体水平抑制心脏衰老。
本发明的研究表明，在心脏细胞衰老过程中，激活TGF-β信号通路可以导致细胞基因组上表观遗传修饰H4K20me3减少，导致基因组的不稳定和基因组损伤修复障碍，从而促进衰老，这一过程可以由氧化应激所引发。进一步研究证明TGF-β/H4K20me3这一衰老通路存在于个体心脏细胞中。TGF-β信号分子细胞膜上受体抑制剂Repsox(E-616452)能够阻断TGF-β信号，抑制了这条衰老通路的激发，从而可以恢复H4K20me3水平并改善衰老小鼠的心脏功能，在老年小鼠中维持年轻心脏的功能特征。从衰老的触发到响应信号的分子通路是一个连续调节的过程，同时伴随着特定的表观遗传学改变，这为治疗心脏老化提供了一个潜在的干预位点。TGF-β信号受体作为靶标，其抑制剂能够应用于预防或延缓细胞衰老和/或心脏老化的药物中。
基于上述发现，本发明提供了用于预防或延缓心脏衰老的方法和药物。阻断或抑制TGF-β信号通路可预防或延缓心脏衰老，其中TGF-β可以是TGF-β1、TGF-β2和/或TGF-β3。通过TGF-β抑制剂可实现TGF-β信号通路的阻断或抑制。所述TGF-β抑制剂可以是抑制活性TGF-β，抑制TGF-β受体，抑制负责活化前体TGF-β为成熟TGF-β的蛋白酶，抑制TGF-β的表达等，或者前述抑制作用的组合。所述TGF-β抑制剂可以是阻断或抑制TGF-β信号通路的任何物质，包括小分子、抗体、蛋白、肽或核酸，其中所述小分子优选为抑制TGF-β受体的小分子，所述蛋白例如蛋白酶，所述核酸例如反义寡核苷酸等。
TGF-β细胞膜上的受体有I、II、III型三种形式：I、II型TGF-β受体均为糖蛋白，而III型受体是一种蛋白聚糖。其中具有信号转导能力的为I和II型。TGF-β受体中I型受体又是将信号传递到细胞内的最重要的受体分子，它能够磷酸化下游的转录因子，从而导致转录因子被激活并锚定到基因的启动子上启动基因转录。
在本发明的具体实施方式中，所使用的TGF-β抑制剂是抑制TGF-β膜受体的小分子Galunisertib(LY2157299)以及Repsox(E616452)。Galunisertib和Repsox是TGF-β受体中I型受体选择性抑制剂，它能够抑制TGF-β受体磷酸化激活，从而抑制了TGF-β信号传入细胞内。在本发明的具体实施方案中，我们发现用Repsox喂养小鼠能够在心脏中抑制TGF-β/miR-29这一衰老通路，导致小鼠心脏在年龄的增长中，能够很好地保持年轻小鼠心脏功能状态。
本发明所揭示的TGF-β/H4K20me3这一表观遗传衰老通路还从未发现过。本发明阐明了TGF-β信号通路能够控制表观遗传修饰H4K20me3水平进而调控衰老进程，进而证明这条TGF-β/H4K20me3衰老通路特异地存在于心脏中。更为重要的是，体内体外实验证实衰老信号分子为TGF-β，通过抑制TGF-β衰老信号膜受体可干预心脏衰老过程。
附图说明
图1显示TGF-β信号促发细胞衰老。其中：a，与对照细胞相比，在体外培养的鼠成纤维细胞中加入蛋白TGF-β1，TGF-β2以及TGF-β3会导致衰老细胞的标记信号β半乳糖苷酶染色信号更早的出现，而TGF-β抑制剂LY2157299以及E616452(Repsox)的加入则使得这些衰老标记信号消失；b，左图显示TGF-β抑制剂LY2157299以及E616452(Repsox)可以增强细胞增殖能力标志蛋白Mki67的标记信号，右图为统计分析柱状图。
图2显示细胞衰老过程中发生表观遗传修饰H4K20me3水平下调。其中：a，细胞衰老过程中表观遗传组蛋白修饰H4K20单甲基化、双甲基化以及三甲基化水平都逐渐丢失，尤其是H4K20me3修饰水平急剧下降，这种情况在高氧培养细胞条件下(20％)更为明显；b，当加入蛋白TGF-β1，TGF-β2以及TGF-β3会导致H4K20me3水平降低以及TGF-β下游转录因子Smad2磷酸化水平上升，而TGF-β受体抑制剂LY2157299以及E616452(Repsox)的加入会导致H4K20me3修饰水平的维持以及TGF-β下游转录因子Smad2磷酸化水平下降。
图3显示表观遗传修饰H4K20me3水平下降促发细胞衰老发生。其中：a，敲低H4K20甲基化酶Suv4-20会显著降低细胞中H4K20二甲基化和三甲基化水平；b，通过敲低H4K20甲基化酶Suv4-20h降低细胞中H4K20甲基化水平会导致细胞增殖生长抑制；c，敲低H4K20甲基化酶Suv4-20会增强细胞衰老相关β半乳糖苷酶的表达；d，敲低H4K20甲基化酶Suv4-20减弱细胞增殖能力标志蛋白Mki67的标记信号。
图4显示在个体衰老过程中心脏细胞存在TGF-β/H4K20me3衰老通路。取1个月、2个月、1年、1.5年以及2年的小鼠中分别从心脏、肾、肝脏、脾以及肺组织细胞进行H4K20甲基化水平检测。心脏细胞随着小鼠个体年龄的增长其H4K20甲基化水平稳定地逐渐降低(a)，而在肾脏中则无明显变化(b)；在肝脏中，年轻小鼠H4K20甲基化水平逐渐上升，但在1年以后的小鼠中则逐渐下降(c左图)；在脾脏和肺组织细胞中则随着年龄的增长，H4K20甲基化水平略微下降或基本不变(c中、右图)。
图5显示TGF-β信号受体抑制剂处能够抑制心脏衰老。其中：a，1岁和2岁小鼠心脏的H4K20甲基化的Western blot(喂或没有喂食抑制剂E-616452)，表面TGF-β膜受体抑制剂能够维持心脏细胞H4K20甲基化水平；b，超声心动图成像测量M-mode和多普勒分析TGF-β受体抑制剂E-616452处理前后心脏功能变化情况，VisualSonics Vevo 2100成像系统及软件(M-mode，左侧和多普勒成像，右侧)展示了1岁小鼠的心脏功能(喂或没有喂食TGF-β抑制剂E-616452)，其中EF代表左侧心室流动的射血分数；E/A代表峰值早期充盈(E波)/舒张晚期充盈(A波)。
具体实施方式
下面结合附图，通过实施例详细阐述本发明。应当理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法和技术，通常按照所属领域的常规条件或按照制造厂商所建议的条件进行。下述实验方法所用的试剂，如无特殊说明，均为自常规生化试剂公司购买得到。
一、材料与方法
1.原代小鼠胚胎成纤维细胞(MEFs)的获取及细胞培养
从12.5-14天大的C57BL/6小鼠胚胎中分离的原代MEFs在DEME培养基(GIBICO，Grand Island，NY，USA)中培养，补充10％胎牛血清(FBS，GIBCO，Grand Island，NY，USA)和1％青霉素/链霉素(GIBICO，GrandIsland，NY，USA)，孵育在37℃、5％CO2的湿化培养箱中。请注意，培养皿在原代HUVEC接种之前用明胶包被。为了建立复制型衰老模型，铺满的MEFs均匀地转移到新的培养皿中，培养细胞直至再次铺满达到群体倍增。细胞培养在100毫米培养皿中，在本研究中，细胞铺满意味着80-90％的生长密度而没有接触抑制。群体倍增数(PD)的数量(n)用公式(n＝log2Ne/Ns)计算，其中Ne和Ns分别是培养结束时的细胞数和开始接种时的细胞数。群体倍增数(PD，倍数)，倍增时间(DT，天数)和总培养时间每3天监测1次，直到MEFs完全停止增殖。
2.衰老相关的β-半乳糖苷酶(SA-β-gal)染色
SA-β-gal活性作为衰老细胞的经典生物标记，也在上述MEFs的所有生长阶段同时进行监测。从6孔板中移去培养基，细胞用1ml的1×PBS清洗一次，用0.5ml固定液在室温下固定10-15分钟。细胞在固定时，根据制造商的说明准备染色液混合物(染色液，染色补充物和DMSO中20mg/ml的X-gal)(Sigma，CS0030，美国)。用1ml的1×PBS清洗两次，用上述染色混合物在37℃孵育细胞过夜。用10×10放大倍率的倒置显微镜观察细胞(莱卡DMI 6000B，莱卡，德国)。用Image J软件(NIH)分析SA-β-gal信号。
3.实时荧光定量PCR(RT-qPCR)
细胞达到80％铺满状态时收获细胞。根据生产商提供的手册用TRIzol试剂(Invitrogen，15596018，USA)分离总RNA。总RNA用随机六聚体引物逆转录成cDNA，用Gapdh标准化的特异引物进行qPCR(LightCycler，Roche，Swiss)测定mRNA、pri-miRNA和pre-miRNA的cDNA水平。至于成熟的miRNA，则按照制造商的说明书(天根，KR211-02，中国)逆转录成cDNA进行qPCR。
4.蛋白质免疫印迹(Western blotting)
总蛋白溶解在1％SDS中，进行SDS-PAGE电泳，转移到硝酸纤维素膜上并用相应的一抗在4℃温育过夜。使用的一抗是抗H3K4me3(Millipore，04-745)，抗H3K9me1(Abcam，ab9045)，抗H3K9me2(Abcam，ab1220)，抗H3K9me3(Abcam，ab8898)，抗H3K9Ac(Millipore，07-352)，抗H3K27me3(Millipore，07-449)，抗-H3K36me2(Abcam，ab9049)，抗H3K36me3(Abcam，ab9050)，抗H4K16Ac(Millipore，07-329)，抗H4K20me1(Abcam，ab9051)，抗H4K20me2(Abcam，ab9052)，抗H4K20me3(Abcam，ab9053)，抗H3(Abcam，ab1791)，抗H4(Abcam，ab10158)，抗Suv4-20h1(Abcam，ab118659)，抗Suv4-20h2(Abcam，ab91224)，抗β-肌动蛋白(Santa cruz，sc-47778)，抗p16(Santa cruz，sc-1207)，抗p15(Santa cruz，sc-612)，抗p21(Abcam，ab109199)，抗Smad2/3(CST，#3102)，抗Smad4(Abcam，ab40759)，抗γH2AX(CST，#9718P)，抗Gapdh(ZSGB-BIO，TA-08)和抗α-微管蛋白(Sigma，T9026)。将蛋白印迹用PBST清洗三次(1×PBS+0.1％Tween-20)，用稀释的二抗(IRDye800CW山羊抗小鼠(LI-COR，926-32210)在室温下孵育2小时。孵育完成后再用PBST清洗三次，然后用奥德赛红外成像系统(Odessey，LI-COR)显影。
5.免疫荧光显微镜
将细胞铺在100mm培养皿中干净的盖玻片上。当达到70-80％铺满状态时，将载玻片上的细胞用冰冷的1×PBS清洗两次，然后用4％多聚甲醛在室温下固定10分钟。室温下用1×PBS清洗细胞3次后，将盖玻片转移到湿润的培养皿中。然后用1％BSA在室温下封闭细胞30分钟。用1×PBS清洗细胞3次后，加150μl一抗在4℃孵育过夜，一抗用PBST(1×PBS+0.1％吐温20)稀释。使用的一抗是抗Ki67(Abcam，ab15580)，抗γH2AX(CST，#9718P)，抗53BP1(Abcam，ab36823)。用PBST清洗细胞4次(1×PBS+0.1％TritonX-100)，然后用150μl二抗(Alexa Fluor 488驴抗小鼠或Alexa Fluor 594驴抗兔，生命技术)在室温下孵育2小时，二抗用PBST(1×PBS+0.1％吐温20)稀释。用PBST(1×PBS+0.1％Triton X-100)清洗细胞3次，并用1ng/μl的DAPI在室温下孵育3分钟。用1×PBS清洗细胞3次，再用ddH2O清洗一次，然后用10μl Fluoromount-G封片剂密封盖玻片，1小时后在荧光显微镜下观察。用Image J软件(NIH)分析免疫荧光信号。
7.染色质免疫沉淀反应
用1％多聚甲醛将大约1.2×106个MEFs在室温下交联10分钟。加入0.125M甘氨酸处理5分钟终止多聚甲醛反应，收集细胞，用细胞核裂解缓冲液(50mM Tris-Cl，10mM EDTA，1％SDS和蛋白酶抑制剂)裂解细胞后，用Bioruptor(Diagenode，比利时)进行超声处理(冰上操作)，用于染色质免疫沉淀实验。用4μg抗Smad4抗体(Abcam，ab40759)或抗兔IgG(Abcam，ab171870)免疫沉淀染色质片段(25μg)。免疫沉淀的DNA通过实时定量PCR进行定量分析，计算免疫沉淀物中的DNA与输入染色质的比例。本分析中使用的引物序列可根据具体要求使用。
8.慢病毒包装和病毒转导
为了生产需要的慢病毒，将编码shRNA靶向Suv4-20h1、Suv4-20h2的pLKO.1-shRNA或Smad4(TRCN文库，Sigma)和两个辅助载体psPAX、pMD2.G共转染到HEK293T细胞中。转染18小时后，将培养基替换为补充有30％FBS的DMEM，24小时后收集病毒上清液。用加入5μg/ml聚凝胺的包装病毒感染细胞(M&C基因技术，MC032，中国)。感染36-48小时后，加入3μg/ml嘌呤霉素处理(M&C基因技术，MA009，中国)24-36小时对细胞进行筛选，然后铺到不同的培养皿中。用1μg/ml嘌呤霉素连续选择4-5天直到在培养皿中无法发现死亡细胞。
9.核小体DNA准备
用1×PBS收集MEFs，用裂解缓冲液(15mM Hepes(pH7.4)85mM KCl和0.5％NP-40)重悬细胞，上述过程中加入了蛋白酶抑制剂混合物(Roche)和PMSF，沉淀细胞核。细胞核沉淀用MNase消化缓冲液(320mM蔗糖，50mM Tris·HCl(pH7.5))4mM MgCl2，1mM CaCl2，0.1mMPMSF)清洗并重悬。为了分离核小体，每0.1mlMNase消化缓冲液中加入0U，0.5U和2U MNase进行消化。37℃下反应2分钟，加入EGTA至终浓度为20mM终止反应。消化产物用RNase在37℃下处理30分钟，然后用蛋白酶K处理并进行两次苯酚氯仿抽提。向上清液中加入3M NaAc(pH5.2)(Na+终浓度为0.3M)，40μg糖原和两倍体积的无水乙醇，提取DNA。沉淀的DNA通过琼脂糖凝胶电泳及溴化乙锭染色进行分析。
10.细胞集落存活检测
将500个细胞一式三份铺在100mm培养皿中，随后用依托泊苷处理至不同的终浓度6小时。6小时后用不含药物的新鲜培养基培养，细胞生长12天后，用1×PB清洗细胞，用4％甲醛固定15分钟，并用结晶紫(0.1％wt/vol)染色30分钟。统计每个培养皿中的克隆数。11.数据分析
通过双尾不成对Student t检验(GraphPadPrism software,version 5.01)或Dunnett多重比较单因素方差分析对数据进行分析，数据表示为平均数±标准差表示。P<0.05时认为有统计学意义。
实施例1.TGF-β信号可以诱导细胞衰老而其抑制剂可以抑制细胞衰老
鼠成纤维原代细胞(MEF)在体外培养的过程中，约进行细胞复制14代左右就会慢慢停止了细胞周期，进入细胞复制性衰老状态。在这个过程中，两个公认的细胞衰老指标会确认这一过程，即衰老相关的β半乳糖苷酶表达逐渐增强，而与细胞复制相关的蛋白Mki67的水平会逐渐下降。我们实验发现TGF-β信号参与了鼠成纤维细胞复制性衰老进程。我们分别在细胞培养基中加入TGF-β蛋白及其细胞膜受体抑制剂，进而观察细胞的衰老状况。我们发现，与对照细胞相比，在体外培养的鼠成纤维细胞中加入蛋白TGF-β1，TGF-β2以及TGF-β3会导致衰老细胞的标记信号β半乳糖苷酶染色信号更早的出现，而TGF-β抑制剂LY2157299以及E616452(Repsox)的加入则使得这些衰老标记信号消失(图1中a)。可见，培养基中加入TGF-β蛋白分子明显促进衰老，而加入TGF-β受体抑制剂则抑制衰老。同时，TGF-β受体抑制剂LY2157299以及E616452(Repsox)也会增强细胞增殖能力标志蛋白Mki67的免疫荧光信号(图1中b)，暗示TGF-β抑制剂可以促进细胞的增殖能力。由此可以看出，TGF-β信号促进了细胞衰老进程，而其受体抑制剂则可以抑制这一进程。
实施例2.TGF-β信号导致基因组表观遗传修饰H4K20me3水平降低
发现TGF-β信号可以促发衰老，我们接着研究这个信号进入细胞内会引起何种表观遗传修饰的改变。我们首先进行了细胞衰老过程中组蛋白修饰变化情况的探索。最终我们发现，在体外培养MEF细胞中，无论是低氧培养条件下(细胞培养氧浓度为3％时减缓细胞衰老进程)还是高氧培养条件下(细胞培养氧浓度为20％时加速细胞衰老进程)，组蛋白修饰H4K20的单甲基化(H4K20me1)，双甲基化(H4K20me2)以及三甲基化(H4K20me3)的水平在衰老过程中都发生了下调，尤其是H4K20me3的下降更为明显(图2中a)。这个结果揭示MEF细胞衰老过程中伴随着组蛋白H4K20甲基化修饰的丢失。加入TGF-β蛋白促进H4K20甲基化修饰的丢失，并且TGF-β信号下游转录因子Smad2的磷酸化水平明显上升，表明TGF-β信号通路在细胞衰老过程中确实被激活(图2中b)。更为重要的是，加入TGF-β膜受体抑制剂LY2157299以及E616452(Repsox)降低Smad2的磷酸化水平，且H4K20甲基化修饰水平得以恢复(图2中b)。这些结果说明细胞衰老过程中TGF-β信号通路被激活，激活后的TGF-β信号通路会导致H4K20甲基化修饰的丢失，而TGF-β受体抑制剂会维持H4K20甲基化修饰水平。
实施例3.降低H4K20me3修饰水平会促发衰老
已知细胞衰老过程中TGF-β信号通路被激活，TGF-β信号能够促发衰老，并且能够降低细胞衰老过程中表观遗传组蛋白修饰H4K20甲基化水平降低，我们预测H4K20甲基化水平降低应该会促发衰老。为验证这种甲基化修饰是否真的会促发衰老，我们利用RNA干扰技术分别或同时敲低H4K20二甲基化和三甲基化酶Suv4-20h1和Suv4-20h2的RNA水平，发现降低了细胞内H4K20二甲基化和三甲基化修饰水平(图3中a)。在此基础上，我们验证了通过敲低甲基化酶造成的H4K20甲基化水平降低对细胞衰老的影响。发现H4K20甲基化水平的降低会抑制细胞的增殖(图3中b)，增强衰老信号β半乳糖苷酶染色强度(图3中c)，减弱细胞增殖能力标记Mki67的信号(图3中d)。这些结果说明H4K20甲基化对细胞的增殖和抑制衰老是必须的。
实施例4TGF-β/H4K20me3衰老通路存在于个体内心脏细胞中
以上结果可知，细胞衰老过程中存在着一条TGF-β/H4K20me3衰老通路。这方面的实验结果都是基于体外培养原代细胞中获得的，接下来的一个关键问题是这条通路是否存在于个体衰老过程中？存在于哪种器官衰老过程中？我们对此进行了探索。
我们从1月龄小鼠饲养小鼠直到呈现衰老状态的2年龄小鼠。在这个过程中，我们从1个月、2个月、1年、1.5年以及2年龄的小鼠中分别从心脏、肾、肝脏、脾以及肺组织细胞进行H4K20甲基化水平。检测发现，随着年龄的增长，小鼠心脏组织细胞中H4K20甲基化水平稳定地逐渐降低，表明在心脏(Heart)中存在着TGF-β/H4K20me3衰老通路(图4中a)。而其它脏器中H4K20的甲基化水平或者维持修饰水平不变，或者略微变化，或者呈现不规律的变化。如图4中b和c所示，随着小鼠个体年龄的增长，肾脏(Kidney)中H4K20甲基化水平无明显变化；在肝脏(Liver)中，年轻小鼠H4K20甲基化水平逐渐上升，但在1年以后则逐渐下降；在脾脏(Spleen)和肺(Lung)组织细胞中则随着年龄的增长，H4K20甲基化水平略微下降或基本不变。由此可以证明TGF-β/H4K20me3这条衰老通路存在于心脏衰老过程中。
实施例5TGF-β信号受体抑制剂处理能够维持H4K20me3修饰水平并抑制心脏衰老
既然小鼠心脏组织细胞中存在着TGF-β/H4K20me3衰老通路，那么TGF-β膜受体抑制剂是否会抑制这条衰老通路并随着年龄的增长能够维持心脏保持较好的功能吗？我们首先对1年龄和2年龄的小鼠进行TGF-β抑制剂(E-616452)灌胃处理1个月，然后进行H4K20甲基化水平检测。发现经过抑制剂处理，H4K20甲基化水平明显得到恢复(图5中a)。伴随着H4K20me3的恢复，心脏的功能指标也明显得到改善，如图5中b所示，通过超声心动图成像测量M-mode和多普勒(Dopler)分析，表明年龄相关的心脏功能下降在抑制剂处理的小鼠中被阻断。这些实验结果表明，TGF-β信号受体抑制剂能够维持H4K20me3修饰水平并抑制心脏衰老，从而使得心脏随个体老化的进程能够维持正常的功能。
Claims (1)
1.TGF-β抑制剂在制备用于预防或延缓心脏衰老的药物中的应用，其中所述TGF-β抑制剂是Galunisertib或Repsox。
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