木犀草素通过SERCA2a介导缺血再灌注损伤的心肌保护作用及其分子机制

Myocardial ischemia-reperfusion (I/R) injury, as a common clinical manifestations in patients with coronary artery disease, in which severe cases can lead to death, is currently the clinical focus. SERCA2a is one of the most important regulating factors of cardiac excitation-contraction coupling, and is closely related to heart failure, cardiomyopathy, arrhythmia, but less studied in I/R injury. Recently, increasing number of studies found that SERCA2a expression decreased in myocardial I/R injury, but the exact mechanism is unknown. Our previous study also found that SERCA2a protein level was significantly reduced in rat myocardial I/R model, but rebounded significantly after luteolin pretreatment. However, the mechanism remains to be elucidated. In view of this, chromosome immunoprecipitation, RNA-protein immunoprecipitation, protein - protein immunoprecipitation and other techniques were used to detect SERCA2a transcription activity, mRNA stability and protein stability in HL-1 cells on the condition of sI/R (simulative I/R: hypoxia-reoxygenation) injury and luteolin pretreatment. In all, this project aims to elucidate the regulatory mechanism of SERCA2a and luteolin pretreatment in the process of myocardial I/R and provides a new theoretical basis for the study of myocardial I/R injury.

缺血再灌注（I/R）损伤是冠心病患者常见的临床表现，严重者可导致死亡，是目前临床关注的热点。肌浆网钙ATP酶2a（SERCA2a）是心肌兴奋收缩偶联最重要的调控因子之一，与心力衰竭、心肌病、心律失常等密切相关，但在I/R损伤中研究较少。近年来初步研究发现I/R损伤时SERCA2a表达下降，但具体调控机制不明；我们前期研究亦发现，大鼠I/R模型中SERCA2a蛋白表达显著降低，而用木犀草素预处理后表达明显回升，但机制有待阐明。本课题采用HL-1心肌细胞系，利用染色体免疫共沉淀、RNA-蛋白免疫共沉淀、蛋白-蛋白免疫共沉淀等技术，从转录、mRNA稳定性和蛋白稳定性三个水平，探讨心肌I/R后SERCA2a的具体调控机制以及木犀草素预处理通过何种机制调控SERCA2a从而发挥对I/R后的心肌保护作用，旨在为I/R损伤的研究提供实验依据。

心肌缺血再灌注（I/R）损伤是危及患者生命的常见临床疾患，近年来关于其机制的研究成为了关注的热点。肌浆网钙ATP酶2a（SERCA2a）是心肌兴奋收缩偶联最重要的调控因子之一，但在I/R损伤中研究较少。本研究从SERCA2a的转录活性、mRNA稳定性、蛋白活性三个层次，主要在HL-1心肌细胞水平的I/R损伤模型，利用荧光素酶报告基因、CHIP、钙瞬变、流式细胞仪等技术，研究木犀草素（Lut）通过SERCA2a保护心肌I/R损伤的作用及调控机制。我们首次发现：.（1）Lut 预处理可以通过调控SERCA2a保护心肌I/R损伤；.（2）Lut 预处理可以通过抑制转录因子 HIF-1α、 EGR1 和上调 SP1 来提高 I/R 心肌的 SERCA2a转录活性；.（3）HIF-1α可以与SERCA2a启动子区域HIF-1α反应元件HRE1、HRE2结合，Lut预处理可以通过HIF-1α调节SERCA2a的转录活性；.（4）I/R损伤及Lut预处理不改变SERCA2a mRNA的稳定性；.（5）Lut可以通过p38MAPK、PI3K/Akt通路提高I/R心肌的SERCA2a蛋白活性。. 上述结果证实了我们提出的“木犀草素通过SERCA2a介导I/R心肌的保护作用”假说，为心肌I/R损伤提供了新的理论依据及防治靶点。