AMPK调控血管周围白色脂肪米色化减轻动脉粥样硬化作用机制研究

Perivascular adipose tissue (PVAT) is associated with atherosclerosis (AS). Thus, seeking and finding the intervention targets for AS treatment and drug development from adventitia is hot issues. We found that the beige-like gene and protein expression of PVAT were enhanced and the inflammation-related cytokines were reduced. However, whether AMPK regulate the phenotype of PVAT to alleviate vascular adventitial inflammation improve AS are still unknown. In this study, we proceed in-depth research in vivo and in vitro based on extensive research foundation. We establish the model of experimental AS in ApoE-/-mice and fat transplantation model of carotid artery, treated with AMPK agonists to investigate the effect of AMPK in the phenotype of PVAT, inflammation of PVAT, vascular function, plaque formation from vascular adventitia. Meanwhile, we use primary adipocytes, macrophages, endothelial cells, smooth muscle cells and isolated blood vessels with co-cultivation assay to discuss the mechanisms of regulation of AMPK in PVAT. Verify above hypothesis from both positive and negative evidence in AMPK over-expression and siRNA interference cells. The present study will confirms the influence and molecular mechanism of AMPK in PVAT and provides the theoretical basis and experimental support for AS treatment in adventitia.

血管周围脂肪组织（Perivascular adipose tissue，PVAT）与动脉粥样硬化（Atherosclerosis，AS）相关，从血管外膜途径寻找和发现血管病变干预靶标是AS治疗和药物研发的热点问题。申请者发现：给予AMPK激动剂可提高PVAT米色化基因和蛋白水平，降低炎症相关因子水平。但AMPK调控PVAT改善AS的确切作用和分子机制仍然未知。因此，申请者基于前期大量研究基础，拟在体内、外开展深入机制研究。体内采用脂肪移植AS小鼠模型和肥胖小鼠模型，探讨和阐明AMPK调控PVAT米色化对血管功能、炎症、斑块的影响；体外采用脂肪细胞、巨噬细胞、内皮细胞、平滑肌细胞及离体血管共培养，阐明AMPK调控PVAT改善AS的分子机制，并采用AMPK超表达和siRNA干扰技术，从正反两方面验证。本项目将阐明AMPK调控PVAT对AS影响及分子机制，为从血管外膜干预AS提供重要实验依据。

本项目以血管周围脂肪在动脉粥样硬化中的作用为科学问题。重点研究AMP激活的蛋白激酶(AMP-activated protein kinase, AMPK)如何调控白色脂肪米色化，尤其对血管周围白色脂肪米色化的影响，以及最终对动脉粥样硬化发病的分子机制。本项目采用高脂喂养建立颈动脉脂肪移植ApoE-/-动脉粥样硬化小鼠模型及C57BL6肥胖小鼠模型，体外采用细胞AMPK过表达、SiRNA干扰及AMPK基因敲除脂肪细胞，结合采用透射电镜、流式细胞仪、血管功能研究体系与分子生物学研究技术，深入阐明AMPK调控PVAT脂肪表型对AS的分子机制，验证AMPK在PVAT白色脂肪米色化中发挥的作用和机制。.本研究的结果发现，在C57BL/6肥胖小鼠模型上，AMPK 激动剂能够降低高脂喂养小鼠体重，降低血糖、血脂总体水平；能够减小白色脂肪、管周脂肪细胞体积，减少其脂肪含量；C57BL/6小鼠高脂喂养后白色脂肪和管周脂肪巨噬细胞增多，巨噬细胞促炎因子分泌增多，抑炎因子分泌减少。AMPK激动剂可以改善脂肪组织巨噬细胞相关炎症，促进巨噬细胞有促炎型向抑炎型极化；AMPK激动剂可以通过血管外膜途径改善血管平滑肌功能。在ApoE-/-动脉粥样硬化小鼠模型上，给予AMPK激动剂小鼠附睾白色脂肪及腹主动脉血管周脂肪细胞体积显著减小，同时使附睾白色脂肪及腹主动脉血管周脂肪的UCP1表达明显增加，促进白色脂肪米色化。减少炎症因子NF-κB p65、MCP-1的表达；显著降低附睾脂肪重量，减少腹腔脂肪含量，改善肠系膜小动脉血管内皮功能及减轻肠系膜小静脉血管内炎症反应，减少腹主动脉斑块面积；颈动脉移植模型发现可减少脂肪移植后小鼠颈动脉脂质沉积与斑块的形成。本研究为从血管外膜途径防治和治疗动脉粥样硬化等血管疾病提供重要实验依据。