脯氨酸羟化酶-2（PHD2）在CVB3 诱导的病毒性心肌炎发病中的作用及机制研究

Viral Myocarditis(VMC)has been recognized as a main cause of sudden cardiovascular deaths among young adults and dilated cardiomyopathy(DCM) among older adults. Less is known about the clinical treatment of VMC. Previous studies shown that it is mainly triggered by the innate and adaptive immune response,but the pathogenesis of VMC is still unclarified. Prolyl hydroxylases (PHDs) play an important regulatory role in cardiovascular diseases such as myocardial infarction. Over recent years, increasing evidences discovered that PHDs also serve as an essential regulators of inflammatory and innate immune functions during viral infection. However, whether PHD2 involve in the regulation of viral myocarditis is unclear. In preliminary experiment of this project, we found that recruitment of macrophages to heart was increased in PHD2-deficient (PHD2-/-) mice, and cardiac tissue levels of proinflammatory cytokines were enhanced compared with wild-type(WT) mice infected with CVB3. Therefore, we hypothesized that PHD2 enzymes exert important functions in systemic inflammation and viral myocarditis. In this project, we will delineate a novel function of the PHD2 in pathogenesis of VMC via the methods of immunohistochemical、ELISA and Western blot etc to test the myocardial inflammation and myocardial dysfunction in PHD2-deficient mice and wild type mice infected with CVB3. Furthermore, bone marrow-derived or heart macrophages will be isolated from the mice for detecting the enhanced proinflammatory macrophage activity and macrophage migration. Downstream effectors of PHD2 in macrophage-mediated inflammation also will be tested in this study to clarify the interference of PHD2 in the innate immune response. Focus was placed on the roles of HIF1α and NF-kB, as these are well-described PHD2 targets with a key regulatory function in innate immunity. This study will help elucidate the regulatory role and mechanism of PHD2 in developing viral myocarditis, thus providing a novel theoretical basis and potential therapeutic target in treatment of viral myocarditis.

病毒性心肌炎（VMC）是青少年猝死及扩张型心肌病的主要诱因，临床治疗却极具挑战。研究表明免疫致病是VMC的主要致病因素，但发病机制未完全阐明。脯氨酸羟化酶-2（PHD2）在多种心血管疾病中具有重要作用，近年，陆续发现PHD 也参与一些病毒感染所致的炎症和固有免疫应答调控，但是否参与VMC的发病未见报道。预实验发现CVB3感染PHD2基因敲除（PHD2-/-）和野生型（WT）小鼠后，前者心肌内巨噬细胞浸润明显增多，心肌炎症加重，推测PHD2在VMC中具有调控作用。本项目拟利用PHD2-/--VMC和WT-VMC小鼠模型，通过免疫组化、酶联免疫、蛋白免疫杂交等方法检测心肌炎症、功能损伤、小鼠巨噬细胞促炎及迁移功能、PHD2效应靶分子HIF1α及NF-κB（介导固有免疫应答）信号通路等变化，探讨PHD2在VMC中的作用及对巨噬细胞和固有免疫应答的影响，阐明调控机制，为临床治疗提供新的思路和靶点。

病毒性心肌炎（VMC）是青少年猝死及成人扩张型心肌病的主要诱因，但其病理机制尚未完全阐明。本项目主要利用野生型（WT-VMC）和 PHD2基因敲除（PHD2-/-）的病毒性心肌炎小鼠模型（PHD2-/--VMC），探讨PHD2对病毒性心肌炎固有免疫应答中巨噬细胞的调控作用。通过对比观察不同感染时间心肌组织内巨噬细胞的浸润，明确 PHD2 对巨噬细胞募集的影响；用氯膦酸二钠脂质体清除小鼠体内巨噬细胞，再用 CVB3 感染小鼠检测炎症指标（如促炎细胞因子 IL1β、IL6 及 TNFα 分泌）的变化，明确 PHD2 引起的炎症改变的确是巨噬细胞依赖性的；通过分离小鼠骨髓来源的巨噬细胞（ BMDMs ），对比观察巨噬细胞的促炎、迁移功能，明确PHD2对巨噬细胞功能的影响；通过检测 PHD2 下游的效应靶分子 HIF1α 和 NF-κB （介导固有免疫应答的关键分子）的表达和活性，同时检测炎症细胞因子分泌，明确 PHD2 对 HIF1α/NF-κB 信号途径的影响，探讨调控机制。本研究得到了相关的重要结果：1.证实了PHD2参与病毒性心肌炎发病过程；2. 明确了PHD2对病毒性心肌炎炎症反应的影响；3.探讨了PHD2对病毒性心肌炎固有免疫应答中巨噬细胞的调控作用及机制，即病毒感染触发免疫应答后，PHD2的缺失可促进巨噬细胞向心脏组织的迁移而影响其促炎功能，使固有免疫应答过激不断招募巨噬细胞浸润心脏，加重了炎症反应，改变了 病毒性心肌炎的病理过程。本项目的实施将为病毒性心肌炎的临床诊治工作提供新的思路和靶点。