沉默miR-301b靶向PPARG对心肌缺血再灌注微循环障碍的作用及机制研究

The no-reflow phenomenon results in a higher incidence of adverse clinical outcomes. Studies have suggested that microvascular dysfunction is considered to be a pathophysiologic sign of the no-reflow phenomenon. This is acknowledging that once ‘no-reflow’ occurs, particularly in its most extreme form, it is difficult to reverse and the prevention and cure strategy is difficult to treat effectively. Our previous study demonstrated that the peroxisome proliferator-activated receptor gamma (PPARG) activation may inhibit NF-κB pathways, which protects against microvascular dysfunction induced by myocardial ischemia-reperfusion (I/R) injury. In the current study, candidate miRNA targets for PPARG were screened using a bioinformatics algorithm，PPARG mRNA has a putative miR-301b binding sequence in the 3’ untranslated region (3’-UTR). In preliminary experiment，the expression levels of miR-301b was significantly up-regulated in response to hypoxia/reoxygenation in vitro and I/R in-vivo. Moreover，PPARG protein were down-regulated in neonatal rat ventricular myocytes (NRVMs) transfected with miR-301b mimic, but up-regulated by miR-301b inhibitor compared with control. Therefore, based on the bioinformatics algorithm, preliminary experiment and previous studies of ours, we propose the hypothesis that pretreatment of miR-301b silencing will increase the cardiac PPARG expression, inhibit the NF-κB pathways and protect against from microvascular dysfunction induced by myocardial ischemia-reperfusion injury. In the present study, firstly，we observe the expression levels of miR-301b, PPARG mRNA and protein in response to hypoxia/reoxygenation in vitro and I/R in-vivo. Secondly, we set out to confirm that PPARG is a direct target of miR-301b in the commonly used HEK293 cell line and in cultures of NRVMs. Thirdly, to observe the protective effect of silence miR-301b in microvascular dysfunction induced by myocardial ischemia-reperfusion injury, we will establish animal model of myocardial ischemia-reperfusion injury in adult male Sprague-Dawley rats. The rats received antagomir-301b at a dose of 80 mg/kg body weight through tail vein injection to decrease the cardiac miR-301b expression. Regional ischemia in vivo was performed at 3 days after pretreatment. By using the antagonist of PPARG (GW9662), we also like to prove that the protective effect of silence miR-301b resulted in increased cardiac PPARG expression. The result of the present study will provide new molecular target for the prevention and cure of no-reflow.

无再流现象的病理生理基础是微循环障碍，目前各种防治措施作用有限。我们前期研究发现，过氧化物酶体增殖物激活受体gamma（PPARG）激活可抑制NF-κB表达，改善心肌缺血/再灌注(I/R)微循环障碍。我们预实验发现I/R损伤后miR-301b表达升高，而且miR-301b与PPARG可能存在靶向关系。因此我们推测：沉默miR-301b预处理可通过靶向上调PPARG表达，改善微循环障碍，缓解无再流。并拟进行以下研究：首先，检测miR-301b、PPARG在I/R损伤时的表达。其次，确证miR-301b与PPARG基因的靶向关系。最后，通过静脉注射antagomir-301b预处理致心肌miR-301b表达沉默，3日后构建大鼠心肌I/R模型，观察沉默miR-301b预处理通过靶向上调PPARG对微循环障碍的改善效应，并探讨作用机制。本课题将为研究防治无再流的分子靶点和靶向策略提供新资料。

急性ST段抬高型心肌梗死再灌注治疗后的“无再流现象”导致了严重的不良临床预后，其病理生理基础是微循环障碍。越来越多证据表明，microRNA在缺血再灌注损伤过程中起重要作用。因此，microRNA调控可能为心肌缺血再灌注微循环障碍提供新的治疗靶点。我们前期研究证实，过氧化物酶体增殖物激活受体gamma（PPARG）激活可抑制NF-κB表达，改善心肌缺血/再灌注(I/R)微循环障碍。我们通过成功建立乳鼠心肌细胞缺氧复氧模型及大鼠心肌缺血/再灌注模型，发现在与PPARG基因靶向匹配的候选microRNA中，miR-301b-3p表达水平升高最明显。在细胞水平，通过双荧光素酶报告基因实验以及RT-qPCR检测，证实PPARG是miR-301b-3p靶基因。最后，通过建立大鼠心肌I/R模型，证实低表达miR-301b-3p预处理通过靶向激活PPARG信号通路，抑制NF-κB的激活，降低炎症因子TNF-α、IL-1β、IL-6在心肌组织的表达，缓解炎症反应；进而减少血细胞在微血管的聚集，缓解毛细血管内皮细胞肿胀，改善微血管的灌注，缩小心肌I/R无再流及心肌梗死面积，改善心功能及血流动力学指标。综上，低表达miR-301b-3p通过靶向激活PPARG信号通路，缓解心肌I/R炎症反应，最终改善微循环障碍。因此，miR-301b-3p可能是无再流防治中一个新的基因治疗靶点。