miR-590经由PPARγ/LOX-1通路发挥内皮功能保护作用及其机制研究

Endothelial dysfunction is the trigger of atherosclerosis. It has been confirmed that oxidized low density lipoprotein (ox-LDL) and lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) can induce endothelial dysfunction, while peroxisome proliferator-activated receptor γ(PPARγ) plays a protective role in ox-LDL-induced endothelial injury through inhibiting LOX-1. The mechanism of how PPARγ suppresses LOX-1 is still unclear. We have found that miR-590 targets LOX-1, and PPARγ agonists upregulate the level of miR-590. Thus, we hypothesize that miR-590 can modulate PPARγ/LOX-1 signalling pathway to attenuate ox-LDL-induced endothelial dysfunction. To verify this, we plan on dectecting endothelial function and its related molecules both in vitro cell lines and in vivo mice model. Endothelial cells pretreated with ox-LDL and mice model fed a high-fat diet will be intervened by miR-590 under- and over-expression methods, respecitively. This study will focus on the mechanism of miR-590 regulation of the PPARγ/LOX-1 pathway and the protective role of miR-590 in ox-LDL-induced endothelial dysfunction. The findings may provide new insights to therapy for endothelial dysfunction.

内皮功能障碍是触发动脉粥样硬化的关键环节。已证实凝集素样氧化低密度脂蛋白受体1（LOX-1）与氧化低密度脂蛋白（ox-LDL）结合引发内皮功能障碍，而过氧化物酶体增殖物激活受体γ（PPARγ）则通过抑制LOX-1减轻ox-LDL诱导的内皮损伤，但PPARγ抑制LOX-1的机制尚不明确。我们预实验发现miR-590可下调LOX-1基因表达，而PPARγ激动剂则增加miR-590水平。由此提出假说：miR-590是PPARγ抑制LOX-1的关键因子，PPARγ通过激活miR-590调控LOX-1，进而减轻ox-LDL诱导的内皮功能障碍。为求验证，我们拟构建miR-590高/低表达的ox-LDL诱导内皮细胞和动脉粥样硬化小鼠模型，评估内皮形态功能并检测相关信号分子，旨在明确miR-590在PPARγ调控LOX-1中的关键作用和具体机制，同时验证miR-590对ox-LDL诱导内皮损伤的保护作用。

凝集素样氧化低密度脂蛋白受体1（LOX-1）是在动脉壁内皮细胞表达的主要受体之一，在内皮功能障碍和动脉粥样硬化的发生发展中起着关键作用。最近的证据表明，LOX-1在胰岛素抵抗的条件下表达被上调，并且可以被抗糖尿病药物抑制。我们之前证实了噻唑烷二酮类药物（TZD）对ox-LDL诱导的内皮细胞中的LOX-1具有抑制作用。但是，其潜在机制尚不清楚。通过本项目，我们发现罗格列酮可显著减弱高脂饮食的ApoE-/-小鼠中LOX-1，ICAM-1，VCAM-1，p47phox的表达，延缓动脉粥样硬化病变的发生。在体外实验，我们发现罗格列酮通过调节miR-590-5p抑制LOX-1。罗格列酮显著降低了ox-LDL介导的ICAM-1，VCAM-1和p47phox的表达，而用antagomiR-590-5p预处理细胞后，ICAM-1，VCAM-1和p47phox的表达全部逆转。罗格列酮能够诱导可激活人脐静脉内皮细胞（HUVEC）中的PPAR-γ，并促进其核易位。细胞核内的PPAR-γ通过与其转录启动子区结合而上调miR-590-5p的表达水平。通过在HUVEC中用PPAR-γ⊿NLS质粒转染将PPAR-γ保留在细胞质中则未能激活miR-590-5p。 PPAR-γ启动子区域的突变也降低了miR-590-5p启动子荧光素酶的活性。总之，这些数据表明，PPAR-γ很可能通过内皮细胞中miR-590-5p的转录调控在动脉粥样硬化的治疗中发挥作用。