低剪切力活化IKKε/TBK1-Akt致内皮细胞损伤和动脉硬化的作用及机制研究

Abnormal endothelial shear stress (ESS)via inducing over-inflammation, endothelial cellular injury and proliferation of smooth muscle cells results in atherosclerosis.IKK-related kinases (IKKεand TBK1) participate in the Type I interferon and immune responses which are associated with the release of different cytopines and upregulate nuclear factor (NF)-kB. However, the role of IKKεand TBK1 signalling in the development and progression of atherosclerosis still remains to be underclear. Our previous studies showed that lower ESS exposed to HUVEc was associated with sharp and rapid down-regulation of PI3K/mTORC2-protein kinase B (Akt), followed by the increased phosphorylation of IRF-3, IRF-7 and Akt started from 5 min after exposure to lower ESS. Thus, we postulate that lower ESS induces Akt pathological activation and endothelial injury through inactivating PI3K/mTORC2 and activating IKKε/TBK1-Akt-IRF-3/7 pathways. To test our postulation, endothelial cells, animals with related gene knocked-out and patients with coronary artery disease were used, respectively, and some modern techniques or devices including chronic virus vector transfection, parallel flow chamber, RT-qPCR, Western blot, immunoprecipitation, electron microscope and atomic force microscope were used. The current study will help understanding of the mechanisms underlying the progression of atherosclerosis, and will provide new evidences and additional new clues to development of new drugs targeting at IKKε/TBK1-IRF-3/7 pathways for prevention and treatment of atherosclerosis.

血管局部血流异常致内皮剪切力（ESS）降低是动脉粥样硬化(AS)的主要机制。IKK相关激酶（IKKε和TBK1）介导I型干扰素反应及固有免疫应答，但与AS的关系不明。我们前期研究发现低ESS持续抑制内皮细胞中PI3K/mTORC2、但激活Akt。我们还发现低ESS显著活化核因子(NF)-kB及干扰素调节因子（IRF-3/7）。据此，我们推测低ESS通过激活IKKε/TBK1-Akt/IRF-3/7而损伤内皮细胞。本项目拟采用人脐静脉内皮细胞、动物主动脉弓内皮细胞、基因敲除动物及冠心病患者，运用慢病毒表达载体、平行板流动腔、RT-qPCR、免疫印迹、免疫沉淀、电子及原子力显微镜、血管腔内成像等技术，研究低ESS介导的IKKε/TBK1-IRF-3/7通路促进动AS的作用及机机制，从细胞和整体水平探索低ESS导致内皮功能异常的机制，从而为寻找防治AS的新靶点提供理论及实验依据。

低剪切应力（LSS）在动脉粥样硬化病变和血管炎症的发生发展中起关键作用。既往的研究显示LSS诱导Akt磷酸化水平增加。然而Akt活化的机制及其在LSS诱导的内皮功能障碍中的作用仍不清楚。本课题旨在探究LSS作用下Akt活化的调控机制，并阐明Akt活化在LSS诱导的内皮功能障碍的作用。本项目组围绕研究目标，开展一系列细胞及动物实验，主要研究结果如下：1）Akt在LSS作用下的活化由IKKε调控，且此调节不依赖于PI3K、mTORC2。2）LSS激活的Akt通过调控下游分子IRF3促进内皮细胞炎症。3）LSS通过IKKε显著激活STAT1，促进内皮炎症因子的表达，增加ROS水平，减少NO释放，导致内皮功能障碍。我们的研究结果显示在不同流体模式刺激下，Akt活化的调控机制及其发挥的作用截然不同，此发现为探究LSS导致的动脉粥样硬化性疾病、特别是血管分叉病变的发病机制带来了新机制和新的干预方向。