CN110151973A - 一种生物活性多肽pacap的应用 - Google Patents
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Abstract
本发明涉及一种生物活性多肽PACAP在制备心脏损伤性疾病的预防、治疗或修复药物中的应用。本发明的生物活性多肽PACAP类药物可以显著预防及缓解放射性心脏损伤的毒性，修复放射性心脏损伤的病理改变及心脏功能，为以放射性心肌组织损伤为特征的胸部恶性肿瘤放疗患者提供新型唯一的靶向治疗策略。
Description
技术领域
本发明属于心脏损伤性疾病领域，特别涉及一种生物活性多肽PACAP的应用。
背景技术
放射治疗是肿瘤综合治疗的重要组成部分，约有52.3％的肿瘤患者在其抗肿瘤治疗过程中需要放射治疗。随着接受放射治疗的人数增加和患者总生存改善，放射治疗相关不良反应越来越得到重视，其中放射相关心脏疾病(radiation-related heart diseases，RRHDs)已成为胸部恶性肿瘤放射治疗患者的非肿瘤死亡的主要原因之一。心脏的所有结构,包括心包、心肌、心瓣膜、传导系统和冠状动脉都有可能受到放射损伤。放射相关心脏疾病类型主要包括冠状动脉粥样硬化性心脏病、心包疾病、瓣膜性心脏病、心力衰竭、心律失常、心肌病等。目前RRHDs的干预通常遵循常规心脏疾病患者的治疗标准，而没有针对特异性靶点的预防及治疗药物，很多情况下无法达到预期的治疗效果。目前尚无成熟的预防放射性心脏损伤的药物，有研究表明β受体阻滞剂和ACEI药物对放射性心脏损伤具有预防作用，但是尚无大型III期随机对照临床试验的结果证明。冠心病患者的治疗遵守指南ABCDE，考虑冠脉微循环病变可给予尼可地尔、曲美他嗪等改善微循环药物，部分有指征患者行冠状动脉介入治疗；对于心肌病和心力衰竭的病人，可借鉴的指南治疗原则：1)ABCDE、2)金三角、3)CRTD/ICD，对LVEF明显下降(降幅超过10％)至低于正常值下限(<50％)水平者，若无禁忌，推荐使用ACEI或ARB联合β受体阻滞剂，以延缓心功能不全进展或心衰症状出现；对心包积液患者，可使用非甾类抗炎药及秋水仙碱治疗，积液过多或血流动力学不稳定时，则需考虑心包穿刺。乳腺癌和纵隔淋巴瘤放疗后相关的心脏损伤是报道最多的胸部放疗心血管并发症。研究已证实辐射导致心脏损伤死亡是抵消放疗生存获益的主因，目前尚无有效的临床手段可以预防或逆转放射性心肌损伤及血管粥样硬化。
胸部含有很多危险器官和放射剂量限制组织，在所有放疗相关副作用中，心血管并发症是胸部放射治疗远期非肿瘤死亡的主要原因之一。随着胸部肿瘤治疗疗效的提高，患者生存期延长，放疗引起的并发症尤其是RRHDs和心肌微循环障碍引起了越来越多的关注。目前放射性心脏损伤的治疗通常遵循常规心脏疾病患者的管理标准，而没有针对放射性心脏损伤特异性靶点的治疗药物，由于放射性心脏损伤的潜伏期可长达10-20年，症状发现时多数情况下心脏已有器质性的变化，目前的药物干预无法达到预期的治疗效果，且尚无成熟的预防，治疗和修复辐射所致心脏损伤的药物。所以亟需探究放射性心脏损伤发生的分子机制、放射生物学基础以找到关键的干预靶点，开发药物，预防、修复和治疗放射治疗对心脏的损伤。
发明内容
本发明所要解决的技术问题是提供一种生物活性多肽PACAP在制备心脏损伤性疾病的预防、治疗或修复药物中的应用，可以显著预防及缓解放射性心脏损伤的毒性，修复放射性心脏损伤的病理改变及心脏功能。
本发明提供了一种生物活性多肽PACAP在制备心脏损伤性疾病的预防、治疗或修复药物中的应用。
进一步的，所述药物包括天然人PACAP38、天然人PACAP27或天然人血管活性肠肽VIP的激动剂、类似物、片段或衍生物。
进一步的，所述药物与一种或多种其他预防/治疗剂组合得到药物组合物。
进一步的，所述心脏损伤性疾病包括由一种因素或多种因素引起的辐射损伤或药物损伤。
有益效果
本发明的生物活性多肽PACAP类药物可以显著预防及缓解放射性心脏损伤的毒性(主要通过增加辐照后细胞活力、抑制细胞凋亡及细胞周期阻滞)，修复放射性心脏损伤的病理改变及心脏功能，为以放射性心肌组织损伤为特征的胸部恶性肿瘤放疗患者提供新型唯一的靶向治疗策略。
附图说明
图1A-C为PACAP38对辐射诱导的H9C2大鼠心肌细胞增值的影响；
图2A-E为PACAP38对辐射后H9C2细胞内凋亡相关蛋白的影响；
图3A-E为PACAP38对辐射后H9C2细胞内细胞周期相关蛋白的影响；
图4A-E为PACAP38对辐射后H9C2细胞内NRF2/HO-1信号通路活性的影响；
图5A-K为PACAP38对H9C2细胞辐射损伤转染Nrf2-siRNA后的影响；
图6A-D为PACAP38对C57/BL6J小鼠单心脏照射后21天左心室心脏组织的病理改变；
图7A-B为PACAP38对C57/BL6J小鼠单心脏照射后21天心功能变化。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，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。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
选用PACAP38类药物进行实验验证：
细胞及动物：应用选择了H9C2大鼠胚胎心肌细胞系(细胞来源：ATCC CRL-1446；细胞特点：大鼠心室肌母细胞，可增值传代培养)进行细胞实验研究，选择C57/BL6J小鼠进行动物实验研究。
建立细胞及动物辐照模型：细胞及动物照射的设备为Varian Trilogy直线加速器。建立及完善成熟了细胞照射模型：包括固定、摆位、辐照计划设计、照射实施及照射后继续细胞培养及后续处理等相关技术流程，其中细胞的照射条件为：6MV X线，剂量率3Gy/min，SSD100cm,机臂180°向上，培养皿下放置1cm有机玻璃板作为补偿物，上方放置5cm有机玻璃板作为散射体。同样进一步建立及完善成熟了的小鼠辐照前体位固定、小鼠单心脏辐照计划设计及辐照实施、辐照后小鼠饲养、各时间点心脏标本取材固定处理及小鼠心脏功能检测等相关技术流程。动物的照射条件为：6MV X线，剂量率3Gy/min，SSD100cm，机臂0°向下，照射野1×1cm，小鼠仰卧位，四肢及牙齿固定于有机玻璃板，身体表面覆盖1cm的有机玻璃板作为补偿。根据不同照射剂量对H9C2大鼠心肌细胞及C57/BL6J小鼠的结构及功能影响的预实验结果，对细胞和动物的单次照射剂量分别选择12Gy及14Gy进行研究。
结果：
一、PACAP38对辐射诱导的H9C2大鼠心肌细胞毒性具有保护作用。
1、H9C2细胞照射前2h给予不同浓度PACAP38(10^-7M，10^-9M)预处理，细胞照射剂量为12Gy，细胞照射后48h应用CCK-8法检测细胞生长活力，发现PACAP38可以显著的抑制辐射所致的细胞活力下降(图1A)；
2、应用克隆形成实验检测在不同照射剂量(0,2,4,8Gy)下PACAP38对H9C2细胞克隆形成能力的影响，种植细胞数随照射剂量的增加而增加(500-8000)，结果显示PACAP38促进H9C2细胞的克隆形成能力，并且随照射剂量的增加其保护效应呈放大趋势(图1B、C)。*P<0.05vs.Control group and#P<0.05vs.Irradiation.
二、PACAP38抑制辐射导致的促凋亡相关蛋白BAX表达的升高及抗凋亡相关蛋白Bcl2表达的降低。PACAP38单独作用不引起BAX及Bcl2表达变化。揭示PACAP38保护辐射相关心肌细胞毒性的机制之一是通过抑制辐射导致的心肌细胞凋亡，进一步流式细胞学方法检测细胞凋亡证实了同样结果。照射剂量为12Gy，检测时间点：照射后48h。
1、qRT-PCR检测发现照射后H9C2心肌细胞BAX mRNA表达增加、bcl-2mRNA表达下降，PACAP38预处理可以抑制BAX mRNA表达的增加及bcl-2mRNA表达的下降，此作用在PACAP38高浓度组(10^-7M)尤为显著(图2A、B)；
2、Western blot检测发现PACAP38预处理抑制照射后H9C2细胞BAX蛋白表达的升高，同样，此作用在PACAP38高浓度组(10^-7M)更显著(图2C、D、E)。IR:Irradiation；*P<0.05vs.Control group and#P<0.05vs.Irradiation.
三、PACAP38逆转辐射诱导的H9C2细胞内细胞周期蛋白cyclinB1及CDK1表达下降。PACAP38单独作用不引起cyclinB1及CDK1表达变化。cyclinB1及CDK1是调控细胞周期G2/M控制点的主要蛋白质，提示PACAP38保护辐射相关心肌细胞毒性的机制之一是通过逆转辐射诱导的H9C2细胞内G2/M细胞周期阻滞，流式细胞学方法验证细胞周期也得到同样结果。照射剂量为12Gy，检测时间点：照射后48h。
1、qRT-PCR检测发现照射后H9C2心肌细胞细胞周期蛋白cyclinB1及CDK1mRNA表达下降，PACAP38预处理可以逆转cyclinB1及CDK1mRNA表达的下降，此作用在PACAP38高浓度组(10^-7M)更显著(图3A、B)；
2、Western blot检测发现PACAP38预处理逆转照射后H9C2细胞cyclinB1及CDK1蛋白表达水平的下降，同样，此作用在PACAP38高浓度组(10^-7M)更显著(图3C、D、E)。IR:Irradiation；*P<0.05vs.Control group and#P<0.05vs.Irradiation.
四、照射可以激活H9C2细胞内Nrf2/HO-1信号通路，PACAP38加强辐射后H9C2细胞内NRF2/HO-1表达的进一步增加，但PACAP38单独作用不引起NRF2/HO-1信号分子表达变化。照射剂量为12Gy，检测时间点：照射后48h。
1、qRT-PCR发现照射后Nrf2及HO-1mRNA表达水平较对照组升高，PACAP38照射前2h预处理可以促进照射后Nrf2及HO-1mRNA表达的进一步升高(图4A、B)；
2、Western blot检测发现PACAP38预处理促进照射后Nrf2及HO-1蛋白表达水平的进一步升高。此作用在PACAP38高浓度组(10^-7M)更显著(图4C、D、E)。IR:Irradiation；*P<0.05vs.Control group and#P<0.05vs.Irradiation.
五、H9C2细胞转染Nrf2-siRNA后显著抑制细胞内Nrf2及HO-1的mRNA及蛋白的表达水平，Nrf2-siRNA的干扰效率不受辐射及PACAP38投与的影响，并且Nrf2-siRNA瞬时转染部分消除了PACAP38对H9C2细胞辐射损伤的保护作用。
1、qRT-PCR检测H9C2细胞转染Nrf2-siRNA1(SiNrf2-1)、Nrf2-siRNA2(SiNrf2-2)及Nrf2-siRNA阴性对照(SiNrf2-NC)48小时后细胞内Nrf2及HO-1的mRNA表达水平的变化，发现与阴性对照组相比，SiNrf2-2干扰效率最高，约70％，选择SiNrf2-2作为SiNrf2进行后续实验(图5A、B)；
2、Western blot在蛋白表达水平验证了A、B的qRT-PCR结果，检测时间点为转染后48小时(图5C、D、E)；
3、qRT-PCR检测在照射加或不加PACAP38对SiNrf2转染H9C2细胞后Nrf2及HO-1的mRNA表达水平的影响，SiNrf2或SiRNA-NC转染H9C2细胞后22小时给予PACAP38(10^-7M)预处理，2小时后给予照射，照射后48小时进行检测，发现Nrf2-siRNA的干扰效率不受照射及PACAP38的影响(图5F、G)；
4、Western blot在蛋白表达水平验证了F、G的qRT-PCR结果(图5H、I、J)；
5、CCK8法检测SiNrf2转染对PACAP38保护H9C2细胞辐射损伤的影响(图5K)，SiNrf2或SiRNA-NC转染H9C2细胞后22小时给予PACAP38(10^-7M)预处理，转染后24小时给予照射，照射后48小时进行CCK8检测，发现SiNrf2转染部分消除了PACAP38对H9C2细胞辐射损伤的保护作用，但SiNrf2转染单独作用不影响H9C2细胞活力。NC:Negative Control；IR:Irradiation；*P<0.05vs.Negative Control group and#P<0.05vs.Irradiation ORAccording to the label.
六、PACAP38改善辐照引起的小鼠心肌结构改变及排列紊乱
1、对照组：心肌细胞排列为长条形，细胞核染为深蓝色，细胞质染为红色，心肌细胞排列整齐(图6A)；
2、单独PACAP-38组：心肌结构及排列较对照组无病理性改变(图6B)；
3、单独照射(IR)组：心肌细胞排列紊乱，部分区域心肌细胞结构松散(黑色箭头示空泡样变)(图6C)；
4、照射+PACAP-38组(IR+PACAP-38)：心肌结构形态及排列较IR组有所改善(图6D)。
七、PACAP38显著修复辐照引起的小鼠心功能障碍
C57/BL6J小鼠单心脏照射后21天应用小动物心超平台对小鼠的收缩和舒张功能进行检测，发现各组心脏收缩功能变化不明显，而舒张功能呈现显著改变：
1、与对照组(NC)比较，在辐射暴露后第21天，单纯辐照处理(IR)及辐照联合乳腺癌靶向药曲妥珠单抗处理(IR+TRZ)的并不显著影响小鼠的心脏收缩功能(左室射血分数LVEF)，并且PACAP-38(PAC)对LVEF没有显著影响(图7A)；
2、在辐照后第21天，与对照组比较，单纯辐照处理(IR)及辐照联合曲妥珠单抗处理(IR+TRZ)显著抑制小鼠的舒张功能；同时PACAP-38(PAC)可以有效地修复小鼠的舒张功能障碍。*P<0.05和**P<0.01(图7B)。
Claims (4)
1.一种生物活性多肽PACAP在制备心脏损伤性疾病的预防、治疗或修复药物中的应用。
2.根据权利要求1所述的应用，其特征在于：所述药物包括天然人PACAP38、天然人PACAP27或天然人血管活性肠肽VIP的激动剂、类似物、片段或衍生物。
3.根据权利要求1所述的应用，其特征在于：所述药物与一种或多种其他预防/治疗剂组合得到药物组合物。
4.根据权利要求1所述的应用，其特征在于：所述心脏损伤性疾病包括由一种因素或多种因素引起的辐射损伤或药物损伤。
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