有氧糖酵解参与柯萨奇病毒B3致心肌炎的机制研究

Viral myocarditis (VMC) is a common acquired heart disease in children, but the pathogenesis is remained unknown yet. Previous studies reported that aerobic glycolysis plays an important role in the pathogenesis of the virus infection, and our experiment also found that glycolysis increased in H9c2 cells infected with Coxsackie B3 virus (CVB3). So that we speculated that the glycolysis mechanism is involved in the pathogenesis of CVB3 myocarditis. For this reason, the project will establish a cell model of H9c2 and a mouse model infected with CVB3 to detect glycolysis and glycolytic pathway kinase activity, and gene expressions in the H9c2 cells and in myocardium of the mice, to analyze the glycolysis effect in CVB3 myocarditis. The changes of glycolysis and cell pathology of the H9c2 cell infected with CVB3, and the CVB3 replication will be investigated after inhibition of the rate limiting enzyme of the glycolytic pathway, in order to analyze the role of this pathway on the H9c2 cells infected with CVB3, and the impact of the rate limiting enzymes to the CVB3 life cycle. The changes of glycolysis and associated kinase and related gene expressions in H9c2 cells infected with CVB3 will be observed by inhibiting the expressions of related signaling molecules in PI3K-mTOR-HIF1α pathway, to analysis of the mechanism of that signaling pathway on the glycolysis in H9c2 cell infected with CVB3. Therefore, this research aims to explore the mechanism of glycolysis in the regulation of CVB3 myocarditis, and to provide some new ideas for the prevention and treatment of VMC.

病毒性心肌炎(VMC)的发病机制尚未完全阐明。研究显示糖酵解在部分病毒感染的发病中起重要作用，申请者亦发现柯萨奇病毒B3(CVB3)感染H9c2细胞后糖酵解增加，因而推测CVB3心肌炎有糖酵解机制参与。为此，本课题通过建立CVB3感染的小鼠和H9c2细胞等模型，检测CVB3感染后糖酵解及该途径激酶活性、及基因的表达变化，以分析VMC的糖酵解变化。通过抑制糖酵解途径的相关限速酶，观察CVB3感染H9c2细胞的糖酵解及细胞病理、CVB3复制等变化，以分析糖酵解通路对CVB3感染H9c2细胞的作用及对CVB3生活周期的影响。通过抑制或过表达相关基因以调控PI3K-mTOR-HIF1α通路相关信号分子，观察CVB3感染H9c2细胞糖酵解及相关激酶、相关基因表达的变化，分析该信号通路对H9c2细胞糖酵解的作用机制。因此，本研究将阐明糖酵解参与调控CVB3心肌炎的机理，为VMC的防治疗提供新思路。

本课题通过建立CVB3感染的小鼠和H9c2细胞等模型，检测CVB3感染后糖酵解及该途径激酶活性的表达变化，以分析病毒性心肌炎的糖酵解变化。研究发现，CVB3心肌炎小鼠模型的心肌组织的乳酸值较对照组增高，糖酵解通路相关激酶如HK2、PFKM、PKM2、GLUT4及LDHA等mRNA及蛋白表达增高。CVB3感染H9c2细胞后的细胞病变效应及相应通路蛋白的变化，发现CVB3感染组葡萄糖消耗量及乳酸产生量明显高于对照组；HK2、PFKM、GLUT4等 mRNA和蛋白水平表达上调。加入糖酵解通路抑制剂2DG处理后，CVB3 感染H9c2 细胞不同时间点bcl2/bax mRNA 的表达无明显变化；但不同浓度抑制剂组的Atg5，Beclin1 mRNA的表达显著下调。加入糖酵解通路抑制剂SCT后，抑制剂组在不同时间点对自噬基因的调控有所不同。说明糖酵解机制在CVB3心肌炎的发病中发挥了重要作用。