β-arrestin 2对TLR9介导的心肌缺血再灌注损伤的调控机理

Ischemia cardiomyopathy, resulting in high disability and mortality, is mainly caused by myocardial ischemia reperfusion (I/R)injury. The innate immune response plays an important role in myocardial I/R injury. Discovery of Toll-like receptors (TLRs)— a key issue of innate immune response, exposes a possible target to prevent myocardial I/R injury, which is TLR9.β-arrestin 2 (β-Arr2) modulates TLR and TLR-mediated signaling pathways, thus regulating innate immune response. We have found that β-Arr2 may be involved in regulating TLR9-mediated myocardial I/R injury, however, the functions and mechanisms are still unclear. In this project, we will evaluate the functions and mechanisms of β-Arr2 in TLR9-mediated myocardial I/R injury in vitro and in mouse myocardial I/R injury model, by β-Arr2 over expression or deficiency. Also we will knock out TLR9 in mouse to define the mechanisms by which TLR9 mediate myocardial I/R injury, and by which β-Arr2 regulates TLR9-mediated signaling pathways. Our studies will provide critical information for new approaches for the treatment of patients with ischemiacardiomyopathy.

心肌缺血再灌注损伤是导致缺血性心脏病患者伤残乃至死亡的重要原因，固有免疫应答是其发生的重要机制。激活固有免疫应答的关键分子—Toll样受体(TLRs)的发现，使心肌缺血再灌注损伤暴露了一个可能的干预靶点—TLR9。β-arrestin 2 (β-Arr2) 调控Toll样受体及TLR介导的信号传导通路，参与调控固有免疫应答。我们预实验发现β-Arr2可能参与调控TLR9介导的心肌缺血再灌注损伤，但具体作用和机制不明。本项目将采用过表达和敲出β-Arr2基因，在体外心肌细胞和小鼠心肌缺血再灌注损伤模型中，探索β-Arr2对TLR9介导的心肌缺血再灌注损伤的作用及机制，并通过敲TLR9基因鼠进一步阐明TLR9介导的心肌缺血再灌注损伤的免疫学机制，及β-Arr2与TLR9信号通路的关系与作用。本项目将重点解答β-Arr2能否干预TLR9介导的心肌缺血再灌注损伤的关键问题，为临床防治提供理论指导。

课题以大鼠心肌细胞和C57雄性小鼠为研究对象，建立了心肌细胞缺氧/复氧模型、小鼠心肌缺血再灌注模型，采用基因沉默、基因过表达、基因测序等技术研究β-arrestin 2和TLR9在心肌缺血再灌注损伤中的作用，并阐明其作用的分子机制。实验结果：（1）miR-128-1-5p能够抑制缺氧/复氧或者H2O2诱导的心肌细胞凋亡；同时，作为miR-128-1-5p的直接靶标Gadd45g也能够参与调控缺氧/复氧或者H2O2诱导的心肌细胞凋亡。此外，miR-128-1-5p/Gadd5g可能通过调节p53的表达参与调控缺氧/复氧或者H2O2诱导的心肌细胞凋亡。（2）miR-141-3p能够抑制缺氧/复氧诱导的心肌细胞凋亡；同时，作为miR-141-3p的间接结合的蛋白CHD8也能够参与调控缺氧/复氧诱导的心肌细胞凋亡。miR‑141‑3p和CHD8可能通过调节p21的表达参与调控缺氧/复氧诱导的心肌细胞凋亡。（3）通过基因芯片测序筛选出了一个新的lncRNA，我们命名为lncRNA-HRAT，已经证实能够加剧缺氧/复氧诱导的心肌细胞损伤。综上研究证实miR-128-1-5p、miR-141-3p和lncRNA-HRAT能够通过不同的靶点参与心肌缺血再灌注损伤的过程，我们的研究为心肌缺血再灌注损伤的防治提供了新的理论基础。