Sirt6调控心肌细胞p53及其下游相关氧化应激和死亡信号通路抑制阿霉素心肌损伤的机制研究

Sirt6, a NAD+-dependent nuclear Histone deacetylase, is closely related to energy metabolism regulation and antioxidant stress. Doxorubicin (DOX) leads to cardiotoxicity through the induction of excessive oxidative stress and cell death, which are the crucial mechanisms for DOX injury in hearts. DOX induces a large increase in p53 levels in cardiomyocytes, which play an essential and complex role in the regulation of DOX-induced cardiotoxicity. We have reported that Sirt6 protected hearts against the acute infarction injury by suppressing the excessive oxidative stress. Here, we propose to reveal a new protection of Sirt6 against DOX cardiotoxicity by using an in vivo model induced by the intraperitoneal injection of DOX in Sirt6+/- and WT mice, as well as a DOX-treated cell model in primary cultured neonatal rat cardiomyocytes (NRCMs) with Sirt6 gene silence or overexpression. We further explore the interaction between Sirt6 and p53 in cardiomyocytes by using IP/coIP, EMSA and ChIP experiments, to reveal how p53 recruits Sirt6 to the promoter regions of its downstream pro-oxidative stress proline oxidase and pro-cell death gene Fas, and then reducing H3K9 acetylation levels to suppress the transcription and expression of both proline oxidase and Fas. Furthermore, Sirt6 protects hearts from DOX-induced cardiotoxicity by alleviating oxidative stress injury and cardiomyocyte death. Our study reveals an endogenous protection and innovative negative regulation of Sirt6 on DOX-induced cardiotoxicity, which will provides a new therapeutic strategy for the treatment of DOX-induced cardiomyopathy and offers new findings for oncocardiology.

核去乙酰化酶Sirt6与能量代谢调节和抗氧化应激关系密切。阿霉素（DOX）造成心肌毒性的最基本机制是诱导强烈的氧化应激和细胞死亡。DOX诱导心肌p53大量增加，p53在DOX心肌损伤中起关键和复杂的调节作用。我们拟在Sirt6拮抗氧化应激缓解心脏急性心梗研究的基础上，构建Sirt6+/-小鼠腹腔注射DOX诱导的心脏损伤模型及离体培养的Sirt6沉默和过表达的心肌细胞DOX损伤模型，探讨Sirt6拮抗DOX心肌病的保护作用，利用IP/coIP、EMSA及ChIP等实验技术研究Sirt6与p53的相互作用，阐明p53招募Sirt6至其下游促氧化应激基因脯氨酸氧化酶和促细胞死亡基因Fas的启动子区，减低H3K9的乙酰化状态，抑制这些基因的转录表达，缓解氧化应激反应和细胞死亡，拮抗DOX心脏毒性的内源性保护作用和创新性负性调控机制，为DOX心脏毒性防治和肿瘤心脏病学研究提供新治疗策略和理论依据。

阿霉素（Doxorubicin，DOX）是重要的蒽环抗癌药，但使用过程中很快出现急、慢性毒性，特别是出现严重的呈剂量依赖性的心脏毒性，最终进展到扩张性心肌病、心肌肥厚和心力衰竭。DOX诱发心脏毒性的机制很复杂，目前仍未完全阐明。现已证实心肌细胞死亡是DOX造成心肌病变的最基本机制。Sirtuin 6（SIRT6）是 Sirtuin 家族的一员，主要定位在细胞核，具有烟酰胺腺嘌呤二核苷酸（NAD+）依赖的核糖基转移酶活性和高度特异的组蛋白H3去乙酰化酶活性，与衰老和能量代谢调节密切相关，在多种心脏疾病的发生发展过程中具有重要的调节作用。SIRT6活化缓解心脏肥大、心肌缺血和动脉粥样硬化等疾病，但目前尚不清楚SIRT6调节DOX心脏损伤的作用及分子机制。本项目主要探究SIRT6缓解DOX心脏病的关键性病理变化，即心肌细胞死亡和氧化应激损伤的作用和具体机制。我们采用DOX处理小鼠和离体培养的大鼠乳鼠心肌细胞（NRMCs）成功构建DOX诱导的体内心脏/体外NRMCs损伤模型，观察到DOX心肌病发生时，小鼠死亡增加、体重减轻、心脏萎缩、心脏组织/NRMCs氧化应激水平和心肌细胞死亡增加，血浆/细胞培养液的心肌酶活性增强、心肌纤维化和心功能障碍；同时，心脏组织/心肌细胞SIRT6水平显著降低。与DOX处理的WT小鼠/NRMCs相比较，SIRT6基因敲除/沉默加重DOX诱导的心脏/NRMCs损伤，而SIRT6过表达则显著缓解这些损伤。机制研究方面，发现SIRT6上调心肌细胞内源性的抗氧化系统，并与转录因子p53相互作用，在细胞核中充当p53转录活性的共同抑制因子。p53将SIRT6招募到其下游与细胞死亡调控相关的靶基因Fas和FasL的启动子区域，SIRT6通过降低这两个基因的启动子区域的组蛋白乙酰化水平，进一步抑制DOX活化p53后介导上调的Fas/FasL基因的转录、减少它们的蛋白质表达，进而减弱Fas/Fas L系统的信号传导，抑制其下游Fas-FADD-caspase-8-凋亡和Fas-RIP3-坏死通路的活化，从而减少p53-Fas/FasL信号通路介导的心肌细胞凋亡和坏死，缓解DOX诱导的心脏毒性。本研究首次阐明SIRT6通过与p53相互作用介导创新的表观遗传机制调节心肌细胞的命运，为化疗诱导的心脏毒性损伤防治提供了新的治疗靶点及策略。