Pellino1蛋白及其SUMO修饰在糖尿病心肌病中的作用机制研究

Diabetes mellitus increases the risk of cardiovascular diseases (CVD) and the incidence of heart failure. Diabetic patients can also develop a specific cardiomyopathy called diabetic cardiomyopathy (DCM). DCM has been defined as ventricular dysfunction that occurs independently of coronary artery disease and hypertension. However, molecular mechanisms responsible for DCM are still poorly understood..Diabetes mellitus is characterized by a chronic and low-grade state of inflammation termed as metaflammation which is mainly triggered by metabolic disorder. Metaflammation is regulated by activation of cellular signaling pathways which are shared with the signaling pathways for induction of innate immune and inflammatory responses. Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) play a central role in the induction of innate immune and inflammatory responses by activation of nuclear factor KappaB (NF-κB) signaling pathways. Modulation of TLRs/IL-1R-mediated signaling pathways has been demonstrated to determine the fate of pathophysiologic mechanisms of CVD. Pellino1 interacts with TLRs/IL-1R signaling molecules and regulates activation of NF-κB and mitogen-activated protein kinases (MAPKs) mediated signaling pathways. However, the role of Pellino1 in DCM has not been investigated. Our preliminary data showed significantly increased expression of Pellino1 in the myocardium of DCM mice. The small ubiquitin-like modifier (SUMO) is a conserved factor that post-translationally regulates proteins involved in many cellular functions. We also observed the SUMOylation of Pellino1 was significantly increased in the myocardium of DCM mice. Importantly, inhibition of Pellino1 expression by shRNA silencing of Pellino1 significantly prevented free fatty acid (FFA)-increased caspase-3 activity in cardiomyocytes. We hypothesized that Pellino1 and its SUMOylation play a pivotal role in the pathophysiologic mechanisms of diabetic cardiomyopathy..To critically evaluate our hypotheses, we will determine the role of Pellino1 in the pathogenesis of streptozotocin-induced DCM using cardiac specific Pellino1 conditional gene knockout mice. Furthermore, to elucidate the effect of Pellino1 SUMOylation in its function, We will transfect the myocardium with an adeno-associated virus serotype 9 (AAV9) expressing mutant of Pellino1 SUMO before induction of DCM in mice. Cardiac function and morphologic changes will be measured after induction of DCM. We will also examine the effect of Pellino1 and its SUMOylation on activation of TLRs/IL-1R-mediated signaling pathways using both in vivo and in vitro models that have been established in our laboratory. .These studies will provide an understanding of the signaling pathways that are critical for DCM. It may also be possible to apply this knowledge in a practical fashion to identify new and novel therapeutic approaches to prevent or manage DCM.

糖尿病心肌病（DCM）是导致糖尿病患者发生心衰的主要危险因素。糖尿病时，糖、脂等代谢紊乱可导致TLRs/IL-1R等介导的信号处于激活状态，而Pellino1则是这些信号通路均可参与调控的关键信号分子，但Pellino1是否参与DCM心脏结构与功能的调控尚不清楚。我们的预实验发现：在DCM心肌组织中Pellino1的表达及SUMO修饰增加；采用shRNA沉默游离脂肪酸（FFA）诱导的Pellino1表达，可以抑制心肌细胞凋亡信号caspase-3的活化。我们推测Pellino1作为关键的信号分子可能在DCM的发病机制中起着重要作用。为此，本项目拟从整体动物与体外细胞两个方面，采用Pellino1心肌条件性基因敲除小鼠、AAV9-Pellino1 SUMO位点突变体心肌特异性转染和shRNA干扰等方法，阐释Pellino1在DCM中的分子调控机制和整体功能作用，为临床防治DCM提供新线索。

糖尿病心肌病（DCM）是导致糖尿病患者发生心衰的主要危险因素。糖尿病时，糖、脂等代谢紊乱可导致TLRs/IL-1R介导的信号处于激活状态，而具有E3泛素连接酶活性的Pellino1（Peli1）则是参与该信号通路调控的关键信号分子，但Peli1是否参与DCM心脏结构与功能的调控尚不清楚。本项目从整体动物与体外细胞两个方面，着重验证“Peli1蛋白及其SUMO化修饰是否可通过与TLRs/IL-1R介导的信号通路中的信号分子相互作用，调控下游信号的激活，从而在DCM心肌重塑和心脏功能的调控中发挥重要作用”这一假说。主要包括：①在动态观察发现Peli1表达及其SUMO修饰与DCM心肌结构与功能损伤及TLRs/IL-1R介导的信号转导呈正相关性的基础上，利用Peli1心肌条件性基因敲除小鼠等研究手段，明确Peli1可以作为关键信号分子在DCM心肌重塑中发挥重要作用，而且Peli1可以通过调控TLRs/IL-1R信号通路中的信号分子TRAF6和RIP1的泛素化影响其下游NF-κB信号等的激活，从而影响心肌损伤和心肌纤维化的发生发展；②证实糖尿病所致的脂代谢紊乱-FFA水平升高，可以引起TLR4信号通路的激活，继而引发Peli1蛋白表达及其E3泛素连接酶活性增加，加强信号通路TRAF6蛋白与ECSIT蛋白在线粒体上的相互作用，促使ECSIT由线粒体内膜转移至外膜，抑制线粒体复合物I的活性，导致线粒体失衡，从而在DCM心肌细胞凋亡及早期心肌舒张功能障碍中发挥重要作用；③构建Peli1蛋白SUMO修饰位点突变体，初步阐明Peli1 SUMO修饰对 Peli1蛋白和TRAF6蛋白泛素化修饰的影响。本项目研究成果不仅有助于阐明Peli1蛋白在DCM心肌重塑中的分子调控机制和整体功能作用，为合成能干预Peli1功能的小分子化合物提供关键结构靶点，而且可为进一步深入了解糖尿病脂代谢紊乱在DCM发病中的作用提供新的线索。