木犀草素下调miR-146a/b介导SERCA2a改善心肌缺血/再灌注损伤的作用和机制研究

Myocardial ischemia-reperfusion(I/R) injury is a serious threat to patients with coronary artery disease. As one of the most important regulating factors of cardiac excitation-contraction coupling, SERCA2a plays a crucial role in myocardial I/R injury. Our previous study demonstrated that luteolin could attenuate myocardial I/R injury by upregulating the expression of SERCA2a. We found that luteolin regulated mircoRNA during I/R process according to high-throughput microarray, and identified that miR-146a/b had a close relationship with SERCA2a. However, the mechanism remains to be elucidated. We observed that miR-146a/b could regulate SUMO1 which had the effect of enhancing SERCA2a protein stability through target gene predictive method. But the mechanism also remains unclear. In this study, we use the methods of gene silence and medthylated analysis to investigate the mechanism in two levels of HL-1 cells and gene knockout mouse. We explore further in three aspects：1. The mechanism of luteolin improving myocardial I/R injury is to downregulate miR-146a/b by methylation status in its promoter. 2. The mechanism of miR-146a/b downregulating SERECA2a in posttranscriptional process. 3. The mechanism of miR146a/b downregulating SUMO1 in posttranscriptional process inducing degradation of SERCA2a. In all, this project aims to elucidate the regulatory mechanism that luteolin downregulates miR-146a/b to attenuate I/R injury targeting SERCA2a and SUMO1, which provides a new theoretical basis and drug treatment for the study of myocardial I/R injury.

心肌缺血再灌注（I/R）损伤严重威胁冠心病患者生命，肌浆网钙ATP酶2a（SERCA2a）在心肌I/R损伤中发挥保护作用。 我们前期发现木犀草素（Lut）通过上调SERCA2a改善心肌I/R损伤。通过miR基因芯片我们发现心肌I/R过程中Lut下调miR-146a/b, 其介导SERCA2a表达并调节SUMO1增强SERCA2a稳定性，但机制待阐明。本课题采用HL-1小鼠心肌细胞和基因敲除小鼠，利用甲基化分析、基因沉默等技术，深入探讨：1.Lut如何通过miR-146a/b启动子甲基化下调其表达，改善心肌I/R损伤；2.miR-146a/b怎样在转录后水平下调SERCA2a的表达；3.miR-146a/b在转录后水平下调SUMO1表达，增加SERCA2a降解的机制。阐明Lut下调miR-146a/b介导SERCA2a 改善心肌I/R损伤的机制，为心肌I/R损伤提供理论依据和药物治疗方向。

心肌缺血再灌注（I/R）损伤严重威胁冠心病患者生命，明确心肌I/R损伤的分子机制、寻找有效的干预方法已成为冠心病心肌I/R损伤治疗中一项亟待解决的重要任务。浆网钙ATP酶2a（SERCA2a）在心肌细胞Ca2+调控过程中发挥重要作用。SERCA2a在心肌I/R过程中功能降低可引起细胞舒缩功能减弱，并导致大量Ca2+潴留在胞浆形成钙超载损伤。microRNAs（miRs）参与心肌I/R损伤调控并成为新的干预靶点，并且其在转录后水平调控 SERCA2a 的表达已被证实。我们前期发现木犀草素（Lut）通过上调SERCA2a改善心肌I/R损伤。通过miR基因芯片我们发现心肌I/R过程中Lut下调miR-146a/b介导SERCA2a表达并增强SERCA2a稳定性，但机制待阐明。本课题采用HL-1小鼠心肌细胞和BALB/c小鼠，利用基因过表达及沉默、双荧光素酶基因报告、流式细胞仪、钙瞬变、线粒体膜电位检测等技术，深入探讨Lut下调miR-146b介导SERCA2a改善心肌I/R损伤的机制。我们的研究证实：.(1)Lut预处理可下调miR-146b，改善心肌I/R损伤；.(2)miR-146b抑制SERCA2a的表达加重心肌I/R损伤；.(3)miR-146b抑制SUMO1的表达降低SERCA2a稳定性加重心肌I/R损伤;.(4)Lut可通过减少SERCA2a启动子结合位点HER1甲基化从而调控SERCA2a表达。.上述结果证实了我们提出的“Lut下调miR-146a/b介导SERCA2a2a;改善心肌I/R损伤”的假说，揭示了miR-146b在心肌I/R损伤过程中的作用机制，为其作为药物干预靶点提供实验依据；完善Lut药物改善心肌I/R损伤的作用机制，为其应用于临床心肌I/R损伤的治疗提供更多的实验支持和理论依据。