CN116270715A - Otud5在制备防治心肌缺血再灌注损伤药物中的应用 - Google Patents
Otud5在制备防治心肌缺血再灌注损伤药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域，具体涉及OTUD5在制备防治心肌缺血再灌注损伤药物中的应用。本发明提供了OTUD5在制备保护心脏药物中的理论依据，具体体现在OTUD5在筛选或制备预防、缓解和/或治疗心肌缺血再灌注损伤的药物中的应用，通过确定OTUD5在心肌缺血再灌注损伤(MIRI)中的调控作用，以OTUD5为靶点对抗心肌铁死亡，本发明以此提供了一种OTUD5诱导剂，或其药物制剂，包括能够促进OTUD5表达或活性的药物，用于预防或治疗MIRI，具有十分重要的临床转化价值。
Description
技术领域
本发明属于生物医药技术领域，具体涉及OTUD5在制备防治心肌缺血再灌注损伤药物中的应用。
背景技术
心肌梗死损伤重、危害大，严重威胁人类健康，病因主要为冠状动脉发生粥样硬化性斑块，导致冠脉狭窄、心肌血供明显不足或斑块破裂。及时再灌注是治疗心梗最有效的方式，但也会导致MIRI。MIRI中近50％的心肌梗死面积和再灌注损伤有关。然而，目前临床上仍缺乏针对MIRI的有效干预手段，故探寻MIRI机制中的关键调控分子作为防治靶点具有重要临床意义。
OTUD5是去泛素化酶OTU家族中的成员，将泛素分子从多肽底物上释放，影响底物的泛素降解，增强蛋白稳定性。既往的研究表明OTUD5在许多细胞过程中起关键作用，参与信号传递、转录调控、细胞周期、增殖凋亡、炎症免疫等生命活动的调控。
MIRI导致心肌细胞死亡，干预不及时则影响心功能，进展为心力衰竭。铁死亡是一种铁离子依赖的脂质过氧化物堆积所导致的细胞死亡方式，近年来研究发现，其在MIRI中起重要作用。抵抗心肌细胞铁死亡可有效减小MIRI梗死面积、缓解心功能障碍。
既往研究表明，去泛素化酶可通过调控细胞死亡通路中的底物蛋白稳定性，影响细胞命运。然而，OTUD5是否通过调控铁死亡参与MIRI，用于防治再灌注心肌损伤，尚无研究报道。
发明内容
为解决临床上预防或治疗MIRI仍缺乏有效治疗手段的问题，本发明的主要目的在于提供OTUD5在制备保护心脏药物中的理论依据。通过确定OTUD5在MIRI中的调控作用，以OTUD5为靶点对抗心肌铁死亡，研制能够促进OTUD5表达或活性的药物，用于预防或治疗MIRI。
为了达到上述目的，本发明可以采用以下技术方案：
本发明提供OTUD5在筛选或制备保护心脏的药物中的应用，所述保护心脏以OTUD5单组分试剂或提高OTUD5表达量的试剂作为活性成分。
本发明所述的筛选，指以OTUD5作为药物靶标，通过检测给药前后受试者OTUD5的表达量，其中给药后OTUD5过表达提示为候选药物，此方法也在本发明的保护范围之内。
作为优选方案，所述应用包括OTUD5在筛选或制备预防、缓解和/或治疗心肌缺血再灌注损伤的药物中的应用。
进一步地，所述应用包括OTUD5作为药物靶标或促进OTUD5表达的物质用于制备预防、缓解和/或治疗心肌缺血再灌注损伤的药物。
本发明还提供OTUD5诱导剂在制备保护心脏功能的药物中的应用。
进一步的，所述OTUD5诱导剂包括促进OTUD5基因表达的物质、促进OTUD5蛋白质活性或蛋白质水平的激动剂、或者促进OTUD5的mRNA水平的激动剂、或调控OTUD5表达的miRNA、lncRNA、circRNA等，其促进作用是可逆的或不可逆的；所述促进OTUD5基因表达的物质使OTUD5的表达量提高1倍以上。
更进一步地，所述促进OTUD5蛋白质活性或蛋白质水平的激动剂包括促进OTUD5蛋白质活性或蛋白质水平的蛋白质、多肽、酶、天然化合物、合成化合物、有机物和无机物。
优选的，本发明OTUD5诱导剂包括OTUD5基因片段插入后的腺相关病毒载体，即带有cTnT启动子的重组腺相关病毒AAV9-OTUD5。
本发明还提供一种药物制剂，其特征在于，包括OTUD5诱导剂或单组分的OTUD5，与医药学上可接受的稀释剂或载体结合，所述药物制剂用于预防、缓解和/或治疗心肌缺血再灌注损伤。
本发明使用的“医药学上可接受的稀释剂或载体”包括任何和所有的生理适用的溶剂、分散介质、胞衣、抗菌剂和抗真菌剂、等渗剂或吸收延迟剂等中的一种或多种。医药学上可接受载体的例子包括一种或多种的水、盐水、磷酸缓冲盐水、葡萄糖、甘油或乙醇等及其组合物中的一种或多种。在许多情况下，在该组合物中最好包括等渗剂，例如，糖、诸如甘露醇、山梨醇、山梨醇的多元醇或氯化钠等中的一种或多种。医药学上可接受载体还可以包含少量的辅助物质，例如润湿剂或乳化剂、防腐剂或缓冲液、纳米材料等中的一种或多种，它们增强了本发明以用于制备抗心肌缺血再灌注损伤的药物制剂。
本发明的药物制剂可施用于人类或非人类的其他动物。
与现有技术相比，本发明的有益效果在于：
本发明利用AAV9-OTUD5在小鼠心脏中特异性过表达OTUD5，发现OTUD5过表达能够显著减少急性MIRI，并能改善慢性期心功能障碍和心脏重构；本发明阐明OTUD5能够通过对抗心肌细胞铁死亡从而改善再灌注中急性和慢性心肌损伤；本发明首次揭示OTUD5能够改善MIRI，为再灌注损伤的预防和治疗提供了新的靶点，具有十分重要的临床转化价值。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1是免疫印迹检测小鼠心脏中OTUD5的过表达情况(A)及统计结果(B)。
图2是OTUD5过表达减轻急性心肌缺血再灌注诱导的心肌损伤。A：Evens blue/TTC染色图以及危险区、梗死面积统计结果；B：CK-MB水平；C：LDH水平。
图3是OTUD5过表达减轻急性心肌缺血再灌注诱导的心肌铁死亡。A：铁离子含量；B：MDA水平；C：GSH水平。
图4是心脏超声检测结果显示OTUD5过表达改善慢性心肌缺血再灌注导致的小鼠心功能障碍。A：超声心动图；B：射血分数(EF％)统计结果，C：短轴缩短率(FS％)统计结果。
图5是马松染色和WGA染色实验提示，OTUD5过表达改善慢性心肌缺血再灌注导致的小鼠心脏纤维化，减轻心肌细胞肥大。A：马松染色及统计结果；B：WGA染色及统计结果。
以上各图中，Sham：假手术组；I/R：缺血再灌注手术组；AAV9-Control：注射阴性对照腺相关病毒；AAV9-OTUD5：注射OTUD5过表达腺相关病毒。
具体实施方式
下面通过具体的实施方案叙述本发明。除非特别说明，本发明中所用的技术手段均为本领域技术人员所公知的方法。另外，实施方案应理解为说明性的，而非限制本发明的范围，本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。
实施例1
1实验方法
1.1小鼠缺血再灌注损伤造模
小鼠七氟烷麻醉后置于手术台，胸前消毒，于胸骨左侧第3、4肋间剪开皮肤，钝性分离，打开胸腔暴露心脏，于心耳下缘1mm处打一活结，结扎左冠状动脉的前降支。小鼠30min缺血后松开活结，恢复冠脉血流，缝合伤口。
急性心肌缺血再灌注损伤造模：术前3周尾静脉注射AAV9-OTUD5过表达病毒，缺血30min，再灌24h。慢性心肌缺血再灌注损伤造模：术前1周尾静脉注射AAV9-OTUD5过表达病毒，缺血30min，再灌3周。
1.2腺相关病毒AAV9-OTUD5的构建
腺相关病毒AAV9-OTUD5构建根据OTUD5基因的序列设计PCR扩增引物，引物序列如下所示：
OTUD5-F：GCGATCGCGCCACCATGACTATTCTCCCCAAAAAG
OTUD5-R：ACGCGTTCAACTCTTGTCTGGGGGTG
本发明对所述AAV9-OTUD5的来源没有严格要求，采用常规构建质粒的方法，将上述表达OTUD5基因的片段插入到腺相关病毒载体上，得到带有cTnT启动子的重组腺相关病毒AAV9-OTUD5，所得病毒滴度为6.67×1013(V.g.)/mL。
1.3Evans Blue+TTC染色
小鼠麻醉后进行固定，I/R造模两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，沿主动脉注射伊文氏蓝染料，可观察到小鼠心脏组织变蓝；取下小鼠心脏用PBS清洗，将组织中的线取出，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薄片并放入2％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，第二天取出拍照。
1.4心脏彩超
上述慢性心肌缺血再灌注损伤造模成功后，将四组小鼠分别放在5％七氟烷的麻醉箱中麻醉后，仰卧位固定于操作台上，以1％七氟烷维持麻醉。连接心电图，将心率维持在400-500次/分。应用Vevo3100多普勒超声仪对小鼠进行经胸超声心动图检查，获取小鼠胸骨旁长轴和短轴的M超及B超图像，以评估心脏功能和结构，并计算左室射血分数LVEF(EF％)和左室缩短分数FS(FS％)。
6-8周雄性小鼠随机分为以下4组：(1)假手术+AAV9-Control；(2)假手术+AAV9-OTUD 5；(3)I/R+AAV9-Control；(4)I/R+AAV9-OTUD5。急性心肌缺血再灌注(缺血30min再灌注24h)造模后，检测梗死面积、心肌损伤标志物CK-MB和LDH、以及铁死亡指标。慢性心肌缺血再灌注(缺血30min再灌注21d)造模后，心脏彩超检测心功能、马松染色检测心脏纤维化、WGA检测心脏重构。
2实验结果
通过注射AAV9-OTUD5与AAV9-Control，构建心脏特异过表达OTUD5的小鼠，免疫印迹实验提示OTUD5能被过表达1倍以上(图1)。
Evens blue/TTC染色结果显示，急性心肌缺血再灌注诱发的小鼠心肌梗死面积65％，而OTUD5过表达将梗死面积减少至45％(图2A)。
血清心肌损伤标志物检测结果表明，急性心肌缺血再灌注导致小鼠血清LDH、CK-MB水平升高5-6倍，OTUD5过表达将其减少至3倍左右(图2B)。
生化结果显示，在急性心肌缺血再灌注中，OTUD5过表达减少了40-50％的心肌铁死亡，表现为抑制缺血再灌注诱导的铁离子增加，MDA上升，GSH下降(图3)。
小鼠超声心动图检测结果显示，在慢性心肌缺血再灌注导致的心功能障碍中，OTUD5过表达将射血分数(EF％)从40％提高到55％，短轴缩短率(FS％)从20％提高到30％(图4)。
马松染色和WGA染色实验提示，慢性心肌缺血再灌注导致小鼠心肌纤维化和心肌肥厚，OTUD5过表达可以改善约40-50％(图5)。
Claims (10)
1.OTUD5诱导剂在制备保护心脏功能的药物中的应用。
2.如权利要求1所述的应用，其特征在于，所述应用包括OTUD5诱导剂用于制备预防、缓解和/或治疗心肌缺血再灌注损伤的药物。
3.如权利要求2所述的应用，其特征在于，所述OTUD5诱导剂包括促进OTUD5基因表达的物质、促进OTUD5蛋白质活性或蛋白质水平的激动剂、或者促进OTUD5的mRNA水平的激动剂，其促进作用是可逆的或不可逆的。
4.如权利要求3所述的应用，其特征在于，所述促进OTUD5基因表达的物质包括调控OTUD5基因表达的miRNA、lncRNA、circRNA。
5.如权利要求3所述的应用，其特征在于，所述促进OTUD5蛋白质活性或蛋白质水平的激动剂包括促进OTUD5蛋白质活性或蛋白质水平的蛋白质、多肽、酶、天然化合物、合成化合物、有机物和无机物。
6.如权利要求3所述的应用，其特征在于，所述促进OTUD5基因表达的物质包括腺相关病毒载体，所述的腺相关病毒载体包含OTUD5基因片段。
7.OTUD5在筛选或制备保护心脏的药物中的应用。
8.如权利要求7所述的应用，其特征在于，所述应用包括OTUD5在筛选或制备预防、缓解和/或治疗心肌缺血再灌注损伤的药物中的应用。
9.如权利要求7所述的应用，其特征在于，所述保护心脏的药物以OTUD5单组分试剂或提高OTUD5表达量的试剂作为活性成分。
10.一种药物制剂，其特征在于，包括权利要求1所述的OTUD5诱导剂或单组分的OTUD5，与医药学上可接受的稀释剂或载体结合，所述药物制剂用于预防、缓解和/或治疗心肌缺血再灌注损伤。
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