CN117137907A - Cbr-470-1在制备预防和/或治疗心肌缺血/再灌注损伤的药物中的应用 - Google Patents
Cbr-470-1在制备预防和/或治疗心肌缺血/再灌注损伤的药物中的应用 Download PDFInfo
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Abstract
本发明公开了CBR‑470‑1在制备预防和/或治疗心肌缺血/再灌注损伤的药物中的应用,涉及生物医药技术领域。本发明建立了小鼠心肌缺血/再灌注模型,于造模前进行CBR‑470‑1给药,利用心脏超声、TTC染色、Western blot、DHE染色等实验技术,评估CBR‑470‑1对小鼠心脏功能、内在机制及信号分子的影响,结果发现CBR‑470‑1能够改善心肌缺血/再灌注后小鼠的心功能,减少心脏的梗死面积,减少心肌细胞凋亡及改善氧化应激,提高Nrf2蛋白的表达,改善心肌细胞铁死亡。由此可见,CBR‑470‑1可用于制备预防和/或治疗心肌缺血/再灌注损伤的药物。
Description
技术领域
本发明涉及生物医药技术领域,特别是涉及CBR-470-1在制备预防和/或治疗心肌缺血/再灌注损伤的药物中的应用。
背景技术
缺血性心肌病(ICM)属于冠心病的一种特殊类型或晚期阶段,是指由冠状动脉粥样硬化引起长期心肌缺血,导致心肌弥漫性纤维化,产生与原发性扩张型心肌病类似的临床综合征。随着冠心病发病率的不断增加,ICM对人类健康所造成的危害也日渐严重。缺血性心肌病表现为扩张型心肌病,伴收缩功能损害,是由于心肌长期缺血引起的。
急性心肌梗死(AMI)是一种严重的临床综合征,具有高死亡率和发病率。通过冠状动脉重建术快速恢复血流是急性心肌梗死最有效的治疗方法,但及时再灌注可导致心脏损伤,即心肌缺血/再灌注(I/R)损伤。目前,对这种疾病的还没有足够的治疗方法。
转录因子Nrf2对氧化应激具有重要的防御活性。在静息条件下,Nrf2通过结合Keap1(Cul3泛素连接酶复合物的衔接蛋白)通过泛素化靶向降解。活化的Nrf2蛋白与Keap1解离并易位到细胞核。Nrf2与抗氧化反应元件(ARE)位点结合后,介导几种关键抗氧化酶和细胞保护基因的转录。研究表明,通过遗传或药理学策略强制激活Nrf2级联反应可以保护神经元细胞免受氧化应激。
CBR-470-1是一种非共价的Nrf2激活剂,分子式为C14H20ClNO4S2,化学结构式为:
CBR-470-1通过激活Keap1-Nrf2级联反应,保护SH-SY5Y神经元细胞免受MPP+诱导的细胞毒性。CBR-470-1可以激活SH-SY2Y神经母细胞瘤细胞中的Nrf2级联反应,导致Keap1-Nrf2复合物解离,细胞质Nrf2蛋白稳定和核易位,随后Nrf2途径基因(HMOX1,NQO1和SOD1)的表达增加。
目前关于CBR-470-1在心肌缺血/再灌注损伤中的作用还未见报道。
发明内容
本发明的目的是提供CBR-470-1在制备预防和/或治疗心肌缺血/再灌注损伤的药物中的应用,以解决上述现有技术存在的问题,本发明研究发现CBR-470-1能够改善心肌缺血/再灌注后小鼠的心功能,减少心脏的梗死面积,减少心肌细胞凋亡及改善氧化应激,提高Nrf2蛋白的表达,改善心肌细胞铁死亡,因此,CBR-470-1可用于制备预防和/或治疗心肌缺血/再灌注损伤的药物。
有研究表明,过度氧化应激与心肌I/R损伤的发病机制密切相关。Keap1-Nrf2-抗氧化反应元件(ARE)系统是抗氧化应激的关键防御机制,参与了多种疾病,包括癌症、神经退行性疾病和心血管疾病。Nrf2是一种主转录调控因子,通过与AREs结合,控制各种内源性抗氧化酶的表达,如NADPH:醌氧化还原酶1(NQO1)、超氧化物歧化酶(SOD)、过氧化物还蛋白1(Prdx1)和谷氨酸-半胱氨酸连接酶(Gclc)。氧化还原稳态和保护心功能障碍中发挥着关键作用。有趣的是,Nrf2的活性是由基于泛素Cullin3的E3连接酶Keap1调节的,该酶增加了Nrf2上的多聚泛素链的形成,并随后被蛋白酶体降解Nrf2。越来越多的证据表明,Keap1-Nrf2信号通路可在AMI和I/R损伤后减轻氧化应激并保留心脏收缩功能。因此,本发明通过调节这一途径来改善I/R介导的心脏损伤。
基于此,本发明提供了如下方案:
本发明提供CBR-470-1在制备预防和/或治疗心肌缺血/再灌注损伤的药物中的应用。
进一步地,所述药物通过提高Nrf2蛋白表达水平,抑制心肌缺血/再灌注的氧化应激水平,来发挥预防和/或治疗心肌缺血/再灌注损伤的作用。
进一步地,所述药物通过改善心肌缺血/再灌注后心脏功能,减少心脏的梗死面积和心肌细胞凋亡来发挥预防和/或治疗心肌缺血/再灌注损伤的作用。
本发明还提供一种预防和/或治疗心肌缺血/再灌注损伤的药物,活性成分包括CBR-470-1。
进一步地,所述药物还包括药学上可接受的辅料。
进一步地,所述药物的剂型包括注射剂、颗粒剂、片剂、丸剂或胶囊剂。
本发明还提供CBR-470-1在制备辅助治疗缺血性心肌病的药物中的应用。
本发明还提供CBR-470-1在制备辅助治疗急性心肌梗死的药物中的应用。。
本发明公开了以下技术效果:
本发明公开了小分子化合物CBR-470-1用于制备防治心肌缺血/再灌注损伤的药物的新用途。本发明建立了小鼠心肌缺血/再灌注模型,于造模前进行CBR-470-1给药,利用心脏超声、TTC染色、Westernblot、DHE染色等实验技术,评估CBR-470-1对小鼠心脏功能、内在机制及信号分子的影响,结果发现CBR-470-1能够改善心肌缺血/再灌注后小鼠的心功能,减少心脏的梗死面积,减少心肌细胞凋亡及改善氧化应激,提高Nrf2蛋白的表达,改善心肌细胞铁死亡。由此可见,CBR-470-1可用于制备预防和/或治疗心肌缺血/再灌注损伤的药物。本发明证明CBR-470-1可作为辅助治疗缺血性心肌病及急性心肌梗死的新药物,具有重要的临床指导意义。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为实施例1各实验组小鼠心脏组织的Keap1、Nrf2、SLC7A11和GPX4蛋白表达水平检测结果;其中A为Keap1和Nrf2的WesternBlot检测结果;B为SLC7A11和GPX4的WesternBlot检测结果;C-F分别为Keap1、Nrf2、SLC7A11和GPX4蛋白水平的统计图;
图2为实施例1各实验组小鼠的心脏功能检测结果;其中A为心脏舒缩运动图像;B和C分别为左心室射血分数(EF)和左心室短轴缩短率(FS)比较图;A中标尺为0.1s;
图3为实施例1各实验组小鼠的心脏梗死面积检测结果;其中A为心脏TTC染色的截面图;B和C分别为心肌危险区和梗死区占左室心肌面积的比例;
图4为实施例1各实验组小鼠心脏组织的TUNEL荧光染色显微图(A)以及TUNEL阳性细胞核数百分比对比统计图(B);
图5为实施例1各实验组小鼠心脏组织的DHE荧光染色显微图(A)以及相对荧光强度对比统计图(B);
图6为实施例1各实验组小鼠血清中丙二醛(A)和谷胱甘肽(B)水平检测结果。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1
1.实验动物及饲养
C57BL/6雄性小鼠,8周龄,购自北京维通利华实验动物技术有限公司;分笼饲养,恒温(23-25℃),恒湿(55-70%)。
2.实验分组
动物实验分为4组:健康对照组(Vehicle组)、正常药物组(CBR-470-1组)、模型组(I/R组)和治疗组(CBR-470-1+I/R组),每组各6只;其中,如表1所示,Vehicle组和I/R组小鼠是将C57BL/6小鼠腹腔注射玉米油0.2mL/只;CBR-470-1组和CBR-470-1+I/R组均是用CBR-470-1预处理实验小鼠(腹腔注射含CBR-470-1的玉米油0.2mL/只,CBR-470-1的注射量为10mg/kg),随后对I/R组和CBR-470-1+I/R组小鼠行心肌缺血/再灌注操作。
心肌缺血/再灌注操作的方法参考文献“Y.L.Zhang,P.B.Li,X.Han,B.Zhang,H.H.Li,Blockageoffibronectin1amelioratesmyocardialischemia/reperfusioninjuryinassociationwith activationofAMPLKB1-AMPKsignalingpathway,Oxid.Med.Cell.Longev.2022(2022),6196173(1942-0994(Electronic))”。
表1动物实验分组
3.WesternBlot
使用RIPA缓冲液(#P0013C,Beyotime)从各组的小鼠心脏左心室纯化总蛋白。根据制造商的说明书(#23225,Thermo),使用商业试剂盒测定蛋白质浓度。蛋白(40-50μg)用10%dodecylsulfatepolyacrylamide(SDS)-聚丙烯酰胺凝胶电泳(PAGE)分离,转移到PVDF膜上,然后与一抗混合过夜。用ImageJ软件对所有蛋白条带进行密度分析,每个蛋白条带密度归一化到之前报道的GAPDH水平。4组小鼠心脏组织的Keap1、Nrf2、SLC7A11、GPX4蛋白水平变化以及对比统计图见图1,结果表明I/R后心脏组织中Keap1蛋白水平上升而Nrf2、SLC7A11、GPX4蛋白水平下降;而CBR-470-1预处理后,小鼠心肌组织中Nrf2、SLC7A11、GPX4蛋白水平有所上升而Keap1蛋白水平不变,说明CBR-470-1预处理能够使小鼠心肌缺血/再灌注损伤后的心肌组织中Nrf2、SLC7A11、GPX4蛋白增多。
4.心脏超声心动图评价
小鼠心肌缺血/再灌注操作后24h,对各组小鼠进行心脏超声检测,方法如下:
将小鼠分别放在1.5%异氟烷的麻醉箱中麻醉后,仰卧位固定于操作台上,以1%异氟烷维持麻醉;先找到胸骨旁长轴切面,保存B-mode数据,旋转探头90°获取小鼠心脏左心室胸骨旁短轴切面,胸骨旁短轴切面以左心室乳头肌水平的切面为标志点,用M型超声模式记录下左心室射血分数(EF),左心室短轴缩短率(FS),左心室收缩末期内径(ESD),左心室舒张末期内径(EDD),左心室收缩末期容积(ESV),左心室舒张末期容积(EDV);胸骨旁长轴切面以左心室流出道水平切面为标志点,用B型超声心动图记录室壁运动图像。
检测结果如下:M型超声心动图记录的4组小鼠心脏舒缩运动图像如图2中A所示,显示小鼠I/R后的心脏收缩舒张功能明显下降,而CBR-470-1预处理的模型小鼠,心脏舒缩功能显著改善;图2中B和C依次为4组小鼠EF和FS比较图,表明模型小鼠经CBR-470-1预处理后EF和FS显著升高,CBR-470-1能够有效缓解I/R损伤造成的心功能下降。以上结果显示对心肌缺血/再灌注小鼠提前注射CBR-470-1(2mg/Kg)改善了小鼠的心脏功能。
5.2,3,5-三苯基四氯化唑(TTC)染色
小鼠心肌缺血/再灌注操作后24h,对各组小鼠进行TTC染色,方法如下:
将小鼠腹腔注射0.2mL三溴乙烷过饱和溶液,小鼠麻醉后进行固定,未造模两组(Vehicle组和CBR-470-1组)与I/R造模时一样结扎左前降支,而造模的两组(I/R组和CBR-470-1+I/R组)沿心梗造模肋间开口处,将心脏再次挤出,在上次结扎处再次将心脏左前降支结扎,在心尖处注射0.9mL1%伊文氏蓝染料,让其沿血流流向全身,可观察到小鼠全身变蓝;取下小鼠心脏用PBS清洗,将组织中的线取出,灌胶并将心脏放入-20℃冰箱冷冻定型30min,结束后将心脏放入模具中切成等厚的4个薄片并放入1%TTC染料中,37℃孵箱孵育30min,结束后将心脏切片放入4%组织固定液中固定一晚上,第二天取出拍照。
检测结果如下:4组小鼠心脏TTC染色的截面图如图3中A所示(其中,蓝色为正常区域,红色为缺血区域,白色为梗死区域),与Vehicle组相比,I/R组的梗死区面积显著增大,而CBR-470-1预处理的模型组小鼠的心肌梗死面积明显减小;图3中B和C分别为4组小鼠的心肌危险区和梗死区占左室心肌面积的比例。以上结果显示CBR-470-1预处理可部分挽救I/R损伤所致的心肌梗死面积。
6.TUNEL染色
将4组小鼠心脏取材后予以20%蔗糖浸泡至沉塘,OCTC包埋、-20℃冰冻切片,50℃烤片20min后用甲醇固定10min,PBS清洗并用组画笔在片子上圈出组织,使用0.1%TritonX-100溶液4℃打孔2min,PBS溶液清洗,TUNEL染料37℃孵箱孵育60min,PBS溶液清洗,DAPI染色5min,PBS溶液清洗,将α-actinin染料用PBS稀释100倍,每个组织30μL4℃过夜。第二天复温30min后,将488荧光二抗用PBS稀释100倍,每个组织30μL37℃孵育30min,结束后PBS溶液清洗,用抗荧光淬灭封片剂封片,全程避光操作;用荧光显微镜拍摄荧光亮度,使用ImageJ软件分析。
检测结果如下:图4为4组小鼠心脏组织的TUNEL荧光染色显微镜拍摄图以及4组TUNEL阳性细胞核数百分比对比统计图,结果表明I/R后小鼠的心肌细胞凋亡增多;而CBR-470-1预处理后,小鼠心肌细胞凋亡明显减少,说明CBR-470-1预处理能够使小鼠心肌缺血/再灌注损伤后的心肌细胞凋亡减少。
7.DHE染色
将4组小鼠心脏取材后予以20%蔗糖浸泡至沉塘,OCTC包埋、-20℃冰冻切片,50℃烤片20min后用甲醇固定10min,PBS清洗并用组画笔在片子上圈出组织,使用0.5%TritonX-100溶液打孔15min,PBS清洗,用PBS将DHE染料稀释100倍,每个组织30μL37℃孵育30min,PBS溶液清洗,结束后用抗荧光淬灭封片剂封片,全程避光操作;用荧光显微镜拍摄荧光亮度,使用ImageJ软件分析。
检测结果如下:图5是4组小鼠心脏组织的DHE荧光染色显微镜拍摄图以及4组相对的荧光强度对比统计图,结果显示I/R后小鼠的心肌组织中的氧化应激水平增高,活性氧含量增多;但CBR-470-1预处理后,小鼠心肌的氧化应激水平明显下降,说明CBR-470-1预处理能够减少小鼠心肌缺血/再灌注损伤后的氧化应激。
8.MDA与GSH含量测定
血清中的丙二醛(MDA)和谷胱甘肽(GSH)含量分别用商业试剂盒(#S0131,Beyotime;#A006-2-1,南京建成生物工程研究所)测定,酶标仪检测波长分别为532nm和405nm,试验方法遵循试剂说明书。
检测结果如下:图6是4组小鼠血清中的MDA和GSH含量测定统计图,结果显示I/R后小鼠的血清中的MDA含量增多而GSH含量减少;但CBR-470-1预处理后,小鼠血清的MDA含量显著下降,GSH含量显著上升,说明CBR-470-1预处理能够减轻小鼠心肌缺血/再灌注损伤后的脂质过氧化。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (8)
1.CBR-470-1在制备预防和/或治疗心肌缺血/再灌注损伤的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述药物通过提高Nrf2蛋白表达水平,抑制心肌缺血/再灌注的氧化应激水平,来发挥预防和/或治疗心肌缺血/再灌注损伤的作用。
3.根据权利要求1所述的应用,其特征在于,所述药物通过改善心肌缺血/再灌注后心脏功能,减少心脏的梗死面积和心肌细胞凋亡,来发挥预防和/或治疗心肌缺血/再灌注损伤的作用。
4.一种预防和/或治疗心肌缺血/再灌注损伤的药物,其特征在于,活性成分包括CBR-470-1。
5.根据权利要求4所述的药物,其特征在于,所述药物还包括药学上可接受的辅料。
6.根据权利要求4所述的药物,其特征在于,所述药物的剂型包括注射剂、颗粒剂、片剂、丸剂或胶囊剂。
7.CBR-470-1在制备辅助治疗缺血性心肌病的药物中的应用。
8.CBR-470-1在制备辅助治疗缺血性急性心肌梗死的药物中的应用。
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