基于单核细胞外泌体中miRNA-223的调控探讨丹皮酚抗动脉粥样硬化血管内皮细胞炎症机制

It is an important mechanism of the development of atherosclerosis for circulating blood monocytes participated in the inflammation of vascular endothelial cells . The exosomal miRNAs has been demonstrated to involved in physiological and pathologic processes of atherosclerosis. However, there is little scientific basis for the role of exosomes derived from monocytes to regulate the function of receptor cells.We has found that Paeonol significantly inhibited inflammatory response of atherosclerotic vascular endothelial cells and also regulated monocytes-derived miR-233 via our previously research supported by National Natural Science Foundation of China, but its relevance remains unclarified. Our research group will analyses it from two aspects of in vivo and in vitro, study the function of mechanism of Paeonol on the regulation of monocyte-derived exosomal miRNA-223, reduction of expression of inflammatory target gene of NLRP3 and STAT3, alteration of signal pathway in vascular endothelial cells, inhibition of inflammation of vascular endothelial cells and the formation of plaque via the technology of lentiviral transfection gene silencing gene,signal pathway blocking, luciferase reporter gene detection and so on. Accordingly, The study attempts to reveal the biological mechanism of exosomes in atherosclerosis and to explain the function and fate of exosomal miRNAs when it enter the vascular endothelial cells. Simultaneously, We want to elucidate that the effect of Paeonol on inflammatory transmission between peripheral blood and blood vessels and to provide a certain scientific basis for atherosclerosis from discussion and treatment of toxins theory.

循环血液单核细胞参与血管内皮细胞炎症是动脉粥样硬化的重要机制之一，外泌体所包含的miRNA已被证实参与疾病发生过程，而迄今对单核细胞通过释放外泌体调节受体细胞功能的作用方式知之甚少。课题组已结题国家自然基金项目研究发现，丹皮酚显著抑制动脉粥样硬化血管内皮细胞炎症反应，亦可调控单核细胞的miRNA-223，但其关联性尚未阐明。本课题采用慢病毒转染基因沉默、信号通路阻断、荧光素酶报告基因检测等技术，从体内外两个方面研究丹皮酚调控单核细胞外泌体miRNA-223，下调血管内皮细胞NLRP3、STAT3等炎症靶基因表达及信号通路改变，抑制血管内皮细胞炎症和斑块形成的作用机制。以期揭示外泌体在动脉粥样硬化中的生物学机制；阐述外泌体进入血管内皮细胞后其所包含的miRNA的功能及命运，为探求丹皮酚影响外周血与血管之间的炎性作用的传递及动脉粥样硬化从毒论治提供一定的科学依据。

本项目主要研究丹皮酚（Pae）是否通过调控单核/巨噬细胞（M/MΦ）释放的外泌体miR-223水平减轻血管内皮细胞（,VECs）炎症，发挥抗动脉粥样硬化（AS）作用。主要研究结果如下：.①Pae可以减少高血脂大鼠血清TC、TG含量，降低血清炎症因子IL-1β、IL-6水平。Pae给药后外泌体中miR-223表达升高，并且与Pae处理大鼠来源的外泌体（Pae-exo）共孵育后VECs存活率上升，炎症因子IL-1β、IL-6表达下降，VECs抗性增加、粘附能力降低。表明：Pae可能通过升高外泌体中miR-223含量缓解VECs炎性反应。.②Pae可减轻ApoE-/-小鼠AS斑块的面积；降低IL-1β、IL-6、ICAM-1、VCAM-1水平；抑制ApoE-/-小鼠主动脉M/MΦ的粘附浸润以及主动脉p-STAT3的表达；显著升高ApoE-/-小鼠主动脉miR-223的含量。Pae（24h，60 μM）可以LPS抑制刺激的THP-1细胞的活力；升高LPS刺激的THP-1中miR-223的表达和共培养VECs中miR-223的表达；Pae-exo可降低VEC中IL-1β、IL-6、ICAM-1、VCAM-1的水平及VECs对THP-1细胞的粘附；降低VECs中STAT3，p-STAT3的水平。表明：丹皮酚调控单核细胞外泌体抑制 ApoE -/- 小鼠主动脉斑块病变。.③验证miR-223靶基因为STAT3 mRNA，转染miR-223 mimic后，可以升高泌体和摄取外泌体后的VECs中miR-223的含量，降低STAT3和p-STAT3的表达，抑制IL-1β，IL-6，ICAM-1，VCAM-1的水平及VECs对THP-1细胞的粘附；而miR-223 inhibitor可以逆转Pae的作用，升高STAT3和p-STAT3的表达，提高IL-1β，IL-6，ICAM-1，VCAM-1的水平及VECs对THP-1细胞的粘附。提示：Pae通过升高THP-1源性外泌体miR-223抑制VECs的STAT3通路。. 本项目以外泌体的分泌作为切入点探究Pae外泌体发挥作用的物质基础（miR-223）及其在AS进程中的作用，明确Pae对单核细胞外泌体miR-223 的作用及其对VECs炎症影响的内在联系及分子机制。