GSDMD介导细胞焦亡在阿霉素诱导心脏毒性发病机制中的研究

Doxorubicin (Dox) is a highly effective antineoplastic drug. However, Dox-induced cardiotoxicity limits its clinical application. Inflammatory cell death has been implicated in the pathogenesis of Dox-induced cardiotoxicity. Gasdermin D （GSDMD）executed pyroptosis is a highly inflammatory form of programmed cell death. Our previous study found that Dox could activate GSDMD and pyroptosis in heart tissue and cardiomyocytes. Block GSDMD could attenuate Dox- induced cardiotoxicity. In addition, we demonstrated the direct interaction between Dox and GSDMD-N protein. We hypothesize that 1) Dox produces pyroptosis by directly binding to GSDMD-N,2) GSDMD and pyroptosis play important role in mediating Dox-induced cardiotoxicity and GSDMD may be a new therapeutic target for Dox cardiomyopathy. In this project, we will characterize the molecular model and effect of Dox binding GSDMD-N. We will investigate the effect and mechanism of GSDMD on mediating dox- stimulated cardiomyocytes/macrophages injury. Using GSDMD knock-out mice and myocardial silencing of GSDMD by AAV9 infection, we are going to validate the role of GSDMD in the pathogenesis of Dox cardiomyopathy. These studies will demonstrate the effect and mechanism of GSDMD in Dox-induced pyroptosis and provide a new target and strategy for the treatment of Dox-induced cardiotoxicity.

阿霉素是有效的一线抗肿瘤药物，但它的心脏毒性较大地限制了其临床应用。阿霉素诱导的细胞炎性死亡是引发其心脏毒性的重要原因，据报道，促使细胞焦亡的关键执行蛋白GSDMD是介导细胞炎性死亡的关键因素。我们前期研究发现，阿霉素与GSDMD-N端蛋白存在直接结合，抑制GSDMD活化可以缓解阿霉素心脏毒性。 基于此，我们假设：1）阿霉素可直接结合GSDMD-N端蛋白发挥促焦亡作用；2）GSDMD介导的细胞焦亡在阿霉素心脏毒性中起着重要的调控作用，GSDMD可作为治疗阿霉素性心肌病的新靶点。本项目通过系列分子实验探索阿霉素结合GSDMD-N端的分子模型及其意义，利用系列细胞实验分析GSDMD在阿霉素诱导心肌细胞焦亡中的作用机制，并利用GSDMD敲除小鼠及心肌特异性沉默GSDMD考察其作为治疗阿霉素性心肌病新靶点的可能性。本项目拟揭示GSDMD介导阿霉素诱导细胞焦亡的机制，为治疗阿霉素心脏毒性提供新靶点。

本项目总目标在于考察GSDMD介导心肌细胞焦亡在阿霉素所致心脏毒性中的作用并探索其激活分子机制，为治疗阿霉素性心肌病提供新靶点。包括：(1) 分析细胞焦亡及其执行蛋白GSDMD在阿霉素所致心脏毒性中的介导作用，考察GSDMD作为阿霉素性心肌病治疗和新药开发有效靶点的可能性。(2) 探索阿霉素激活心肌细胞GSDMD的分子机制。.本项目的实施阐明了GSDMD介导阿霉素所致心脏毒性的分子机制，主要包括：1）Dox直接结合GSDMD并促进GSDMD-N调控的焦亡性胞膜打孔；2）炎性Caspases参与调控Dox诱导的心肌细胞焦亡及GSDMD激活；3）GSDMD通过打孔线粒体诱导线粒体损伤参与介导阿霉素心脏毒性。另一方面，Dox以GSDMD依赖的方式诱导心肌细胞发生焦亡；GSDMD基因敲除可以减少Dox诱导的小鼠心肌病。以上发现揭示了Dox诱导的GSDMD激活在其心脏毒性中的关键作用，并强调了GSDMD作为DIC潜在治疗靶点的前景。