CN113702645A - Sirt4在心血管疾病治疗中的用途 - Google Patents
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Abstract
本发明公开了SIRT4在心血管疾病治疗中的用途，属于生物医药技术领域。该用途特别是SIRT4在制备治疗由心肌缺血再灌注损伤导致的急性心肌梗死疾病药物中的用途，包括给受试者的心肌组织施用有效量的所述药物，调节SIRT4的表达从而减轻心肌缺血再灌注损伤。本发明首次提出SIRT4在心血管疾病治疗中的用途，以氧化应激和能量代谢为切入点，分别从动物水平用SIRT4敲除小鼠验证SIRT4在心肌缺血再灌注中的作用和细胞水平用离体心肌细胞低氧复氧损伤模型验证SIRT4在细胞损伤的作用，方法真实可靠，为临床上治疗由心肌缺血再灌注损伤导致的急性心肌梗死疾病提供新的用药途径。
Description
技术领域
本发明属于生物医药技术领域，涉及SIRT4在心血管疾病治疗中的用途，具体涉及SIRT4在制备治疗由心肌缺血再灌注损伤导致的急性心肌梗死疾病药物中的用途。
背景技术
冠心病与急性心肌梗死等心血管疾病是当今世界威胁人类健康的最主要杀手。据2017年中国心血管疾病报告，目前我国急性心肌梗死死亡率总体呈上升态势。通过溶栓或介入治疗及时开通罪犯血管是挽救病人生命的最有效措施。然而，即使接受了优化的血运重建治疗，仍有接近10％的急性心肌梗死病人在出院后年内发生死亡，约20％的存活患者发生慢性心力衰竭，研究表明心肌缺血再灌注损伤是导致心肌梗死患者病死率居高不下的重要原因并成为阻碍血运重建治疗效果的重要瓶颈。缺血再灌注损伤是一个非常复杂的病理生理过程，一方面再灌注治疗是挽救存活心肌的唯一方法，另一方面再灌注促使缺血期存活的心肌细胞发生不可逆性的死亡，在缺血再灌注损伤区域中近50％的心肌梗死面积和再灌注损伤有关，寻找心肌缺血再灌注损伤的关键调节因子作为治疗靶点，对促进心肌梗死优化治疗具有重要意义。
SIRT3、SIRT4和SIRT5作为sirtuins家族的三个主要成员主要在线粒体内表达，并参与线粒体蛋白质翻译后的修饰，已有的研究表明SIRT3和SIRT5参与了心肌缺血性损伤和慢性心力衰竭的发展过程，然而目前尚不明确SIRT4在心脏缺血性损伤发生发展中的作用。既往的研究证明在成纤维细胞和HEK-293T细胞SIRT3蛋白可以调控IDH2蛋白赖氨酸413位点的乙酰化，在SIRT3敲除的心肌细胞或成纤维细胞其IDH2蛋白乙酰化显著增加并且SIRT3敲除小鼠对心肌缺血再灌注损伤的敏感性显著增加，表现为梗死面积和细胞凋亡的增加，心脏功能的恶化。非常有趣的是：在缺血再灌注损伤后，SIRT3敲除的小鼠其心脏的SIRT4蛋白表达显著增加而SIRT5蛋白的表达并未显著改变，进一步的研究采用免疫共沉淀技术证明在心肌细胞SIRT3和SIRT4存在相互作用，然而SIRT4敲除或过表达并未改变SIRT3活性。上述证据暗示SIRT4可能通过调节SIRT3蛋白的脱乙酰作用调控心肌缺血性损伤。
发明内容
本发明的第一目的是提供SIRT4在制备治疗心血管疾病药物中的用途。
本发明的第二目的是提供SIRT4在制备治疗急性心肌梗死疾病药物中的用途。
本发明的第三目的是提供SIRT4在制备治疗由心肌缺血再灌注损伤导致的急性心肌梗死疾病药物中的用途。
根据上述任一所述用途，包括给受试者的心肌组织施用有效量的所述药物，调节SIRT4的表达从而减轻心肌缺血再灌注损伤。
优选地，所述心肌组织包括心肌细胞。
优选地，所述受试者为心肌缺血性损伤患者。
优选地，所述药物为SIRT4抑制剂。
更优选地，所述SIRT4抑制剂为靶向抑制或减少SIRT4表达的小分子、抗体或拮抗性核酸。
与现有技术相比，本发明的有益效果在于：
(1)本发明首次提出SIRT4在制备治疗急性心肌梗死药物中的用途，特别是SIRT4在制备治疗由心肌缺血再灌注损伤导致的急性心肌梗死药物中的用途，以氧化应激和能量代谢为切入点，分别从动物水平用SIRT4敲除小鼠验证SIRT4在心肌缺血再灌注中的作用和细胞水平用离体心肌细胞低氧复氧损伤模型验证SIRT4在细胞损伤的作用，方法真实可靠。
(2)本发明一方面在动物水平采用SIRT4敲除小鼠验证SIRT4在心肌缺血再灌注中的作用，结果表明敲除SIRT4能够显著减轻心肌缺血再灌注损伤，过表达SIRT4能明显加重心肌缺血再灌注损伤；一方面在细胞水平采用离体心肌细胞低氧复氧损伤模型验证SIRT4在细胞损伤的作用，结果表明SIRT4可能通过调节SIRT3蛋白的脱乙酰作用调控心肌缺血性损伤。
参考以下详细说明更易于理解本发明的上述以及其他特征、方面和优点。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点会变得更显著：
图1是不同时间低氧处理促进心肌细胞SIRT4表达的结果。
图2是SIRT4过表达加重小鼠心肌缺血再灌注损伤的结果；其中：(A)SIRT4过表达显著增加心肌梗死面积，虚线标注区域为心肌梗死区(不能被Evans blue和TTC染液染色)，实线标注区域为心肌缺血区(不能被Evans blue但能被TTC染色)，未标注区域为非缺血区(被Evans blue和TTC染液染色)；(B)SIRT4过表达的IR小鼠心肌细胞凋亡显著增加(400×)；(C)SIRT4过表达加重IR小鼠心脏功能障碍；AAV-EGFP：空载病毒对照；AAV-SIRT4：SIRT4过表达；Sham：假手术组；IR：小鼠心肌缺血再灌注损伤组；*P<0.05，每组样本数为6。
图3是过表达及敲除SIRT4验证其对离体心肌细胞存活的影响；其中，Ad-GFP：过表达对照组；Ad-SIRT4：高表达SIRT4组；sh-GFP：敲除对照组；sh-SIRT4：SIRT4敲除组；Control：正常培养；H/R：心肌细胞缺氧再复氧；杆状细胞为正常存活心肌细胞，圆形心肌细胞为死亡心肌细胞。
图4是SIRT4敲除改善小鼠心肌缺血再灌注损伤的结果；其中，(A)SIRT4敲除显著降低小鼠心肌梗死面积，虚线标注区域为心肌梗死区(不能被Evans blue和TTC染液染色)，实线标注区域为心肌缺血区(不能被Evans blue但能被TTC染色)，未标注区域为非缺血区(被Evans blue和TTC染液染色)；(B)SIRT4敲除的IR小鼠心肌细胞凋亡显著降低(400×)；(C)SIRT4敲除改善IR小鼠心脏功能损伤；WT：野生小鼠对照，SIRT4 KO：SIRT4敲除；Sham：假手术组；IR：小鼠心肌缺血再灌注损伤组；*P<0.05，每组样本数为6。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面结合本发明实施例的附图，对本发明实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其它实施例，都属于本发明保护的范围。
以下实施例中涉及的小鼠购自常州卡文实验动物中心，TUNEL分析试剂盒购自碧云天生物科技有限公司，Evans blue和TTC由美国Sigma公司提供，动物实验已通过复旦大学附属中山医院伦理委员会批准。
成年心肌细胞的分离可参见文献(A Simplified,Langendorff-Free Method forConcomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From theAdult Mouse Heart.Circ Res.2016；119:909-920.)。
实施例1构建缺血再灌注损伤动物模型
提前对成年C57BL/6小鼠和SIRT4 KO小鼠进行胸部脱毛处理，使用2％的异氟烷气体吸入麻醉小鼠，行左胸开胸手术，将胸大肌与胸小肌分离，止血钳打开胸膜和心包，暴露心脏使用6-0丝线对其前左降支进行活结结扎缺血40min，随后解开结扎线实施心肌再灌注，再灌注24小时后行超声心动图检测小鼠心脏功能，Evans blue/TTC染色检测小鼠心肌的梗死面积。
Evans blue/TTC染色的具体步骤为：再灌注24小时后，不同处理组小鼠进行腹腔注射1％戊巴比妥钠0.2ml，小鼠麻醉后固定在小动物操作台上，开胸快速暴露心脏，使用6-0丝线重新结扎小鼠前降支，同时暴露左心耳；胰岛素注射器吸取1％伊文思蓝染液注射于左心耳，待心脏未结扎区充满染液；取下心脏，在无菌生理盐水中洗涤三次去除多余染液，去除非心肌组织，并保证左右心室完整。将心脏转移到-80℃冰箱中冷冻30min，随后取出冷冻变硬的心脏，冰上迅速将变硬的心脏横向均匀切成4-5片，心脏切片放置于1％TTC染液中，避光37℃水浴30min后，将心脏压平，固定在4％多聚甲醛中，于体视显微镜下拍照观察。其中，虚线标注区域为心肌梗死区(不能被Evans blue和TTC染液染色)，实线标注区域为心肌缺血区(不能被Evans blue但能被TTC染色)，未标注区域为非缺血区(被Evans blue和TTC染液染色)，梗死面积以危险区的百分比计算。
实施例2验证缺氧与心肌细胞中SIRT4表达水平关系
酶消化法分离小鼠成年心肌细胞，培养在laminin包被的培养皿中，待细胞贴壁后分别进行缺氧处理0h、1h、10h和18h。将处理后的细胞收集后裂解提取蛋白进行Westernblot检测，结果如图1所示，缺氧处理后心肌细胞的SIRT4表达水平明显升高。
实施例3验证SIRT4过表达加重小鼠心肌缺血再灌注损伤
通过尾静脉注射SIRT4(AAV9-CMV-SIRT4-P2A-EGFP-3FLAG)腺相关病毒4周后构建心肌细胞SIRT4高表达小鼠，并建立在体缺血再灌注损伤模型(缺血40min，再灌注24h)再灌注24小时后，分别进行Evans blue/TTC染色，心肌细胞凋亡检测以及小鼠超声心动图检测，发现SIRT4过表达显著增加心肌梗死面积(图2A)，促进小鼠心肌细胞凋亡(图2B)，加重心脏功能障碍(图2C)。
实施例4离体实验正反向验证SIRT4对小鼠心肌功能的影响
酶消化法分离小鼠离体心肌细胞，培养在laminin包被的培养皿中，进行腺病毒转染，转染Ad-SIRT4腺病毒以高表达SIRT4，转染sh-SIRT4腺病毒以抑制SIRT4表达。之后模拟小鼠在体心肌缺血再灌注损伤在体模型，离体建立心肌细胞低氧复氧损伤模型，对心肌细胞的存活率进行分析，结果发现SIRT4敲除显著促进心肌细胞存活，SIRT4过表达降低心肌细胞存活(图3)。
实施例5验证SIRT4敲除改善小鼠心肌缺血再灌注损伤
为验证SIRT4敲除对小鼠心肌的保护作用，分别建立野生(WT)和SIRT4敲除(SIRT4KO)小鼠缺血再灌注损伤模型(缺血40min，再灌注24h)再灌注24小时后，分别进行Evans blue/TTC染色，心肌细胞凋亡检测以及小鼠超声心动图检测，结果表明SIRT4敲除显著降低心肌梗死面积，抑制小鼠心肌细胞凋亡，改善心脏功能障碍(图4)。
Claims (8)
1.SIRT4在制备治疗心血管疾病药物中的用途。
2.SIRT4在制备治疗急性心肌梗死疾病药物中的用途。
3.SIRT4在制备治疗由心肌缺血再灌注损伤导致的急性心肌梗死疾病药物中的用途。
4.根据权利要求1或2或3所述的用途，其特征在于，包括给受试者的心肌组织施用有效量的所述药物，调节SIRT4的表达从而减轻心肌缺血再灌注损伤。
5.根据权利要求4所述的用途，其特征在于，所述心肌组织包括心肌细胞。
6.根据权利要求4所述的用途，其特征在于，所述受试者为心肌缺血性损伤患者。
7.根据权利要求4所述的用途，其特征在于，所述药物为SIRT4抑制剂。
8.根据权利要求7所述的用途，其特征在于，所述SIRT4抑制剂为靶向抑制或减少SIRT4表达的小分子、抗体或拮抗性核酸。
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