基于AMPK/STAT1通路调控巨噬细胞介导的炎症反应探讨益气活血法稳定动脉粥样硬化易损斑块的机制研究

The instability of atherosclerotic plaques which may result in plaque rupture are the main causes of acute cardiocerebrovascular events, and macrophage mediated inflammation is the pathological basis of plaque rupture.Recently studies have shown that AMPK/STAT1 pathway is an important approach to regulate macrophage inflammatory response, and activating AMPK or repressing STAT1 signals can stabilize vulnerable plaques.TCM holds that qi deficiency and blood tasis are the main pathogenesis of AS , and the main therapeutic principle is yiqi huoxue method.Previous studies have found that Buyang Huanwu decoction which is the tipical formula of yiqi huoxue decoction has effects to anti-inflammatory and stabilize vulnerable plaques, but its mechanisms are remain unclear, therefore we propose a hypothesis that the Buyang Huanwu decoction stabilize vulnerable plaques through mediating macrophage inflammatory based on the activation of AMPK/STAT1 pathway. In order to clarify the mechanisms of Buyang Huanwu decoction ,this study intend to use ApoE - / - mice for vulnerable plaques model and ox - LDL induced macrophage for foam cells model, and apply pathway activity analysis, gene silencing, protein function antagonism to evaluate macrophage inflammatory reaction and analyze their correlations of AMPK/STAT1 pathway.The signaling proteins regulated in this research promise to be important targets for stability AS plaques in clinical.

动脉粥样硬化（AS）斑块不稳定乃致破裂是导致急性心脑血管事件发生的主因，而巨噬细胞介导的炎症反应是导致斑块破裂的病理基础。研究表明AMPK/STAT1通路是调控巨噬细胞炎症反应的重要途径，激活AMPK或抑制STAT1信号可稳定易损斑块。中医学认为AS的主要病机为气虚血瘀，益气活血法为其治则。前期研究发现益气活血法代表方--补阳还五汤具有抗炎及稳定斑块的作用，然其机制未明。据此本研究提出假说：益气活血法可通过激活AMPK/STAT1通路，抑制巨噬细胞介导的炎症反应，下调巨噬细胞中促炎因子的表达，最终稳定易损斑块。本研究拟以ApoE-/-小鼠复制易损斑块模型和巨噬细胞复制泡沫细胞模型，采用通路活性分析、基因沉默、蛋白功能拮抗等方法评价巨噬细胞炎症反应与AMPK/STAT1通路的相关性，并阐明补阳还五汤的作用机制，以验证上述假说。本研究所涉及的信号蛋白有望成为临床稳定AS斑块的重要靶点。

巨噬细胞介导的炎症反应是导致易损斑块破裂的病理基础。AMPK/STAT1通路是调控巨噬细胞介导的炎症反应的重要途径，且激活AMPK或抑制STAT1信号可稳定AS易损斑块。“益气活血法”代表方---补阳还五汤在临床广泛应用于AS所致的急性心脑血管疾病且疗效显著。本课题组前期研究发现补阳还五汤具有良好的抗炎作用，据此提出假说：补阳还五汤可通过激活AMPK/STAT1信号，抑制斑块内巨噬细胞介导的炎症反应，下调巨噬细胞中促炎因子表达，最终稳定AS易损斑块。本项目在体以高脂饲养apoE-/-小鼠16周复制AS易损斑块模型，结果发现：补阳还五汤可抑制apoE-/-小鼠主动脉斑块形成，减少斑块内脂质含量、胶原含量及巨噬细胞数，从而稳定易损斑块；补阳还五汤可明显降低apoE-/-小鼠血清TC且升高HDL-C水平，提示补阳还五汤具有一定的调脂作用，可能参与胆固醇逆转运过程；补阳还五汤可明显降低apoE-/-小鼠血清及主动脉中IL-1β、TNF-α、MMP-1、MMP-9、ICAM-1的表达；补阳还五汤可明显上调apoE-/-小鼠主动脉AMPKα1、AMPKα2的蛋白含量及磷酸化水平，且下调STAT1的磷酸化水平。离体以100μg/ml ox-LDL诱导RAW264.7巨噬细胞24h复制泡沫细胞模型，结果发现：补阳还五汤含药血清可呈剂量依赖性地下调ox-LDL诱导的巨噬细胞中TNF-α、MMP-1、MMP-9、ICAM-1的蛋白表达及转录水平；补阳还五汤含药血清可上调ox-LDL诱导的巨噬细胞中AMPKα1、AMPKα2且下调STAT1的蛋白含量及磷酸化水平；Compound C可阻断AMPK对STAT1的抑制作用；Compound C或STAT1过表达干预均可阻断补阳还五汤含药血清对ox-LDL诱导的巨噬细胞TNF-α、MMP-1、MMP-9、ICAM-1表达的下调作用。最终得出结论：补阳还五汤可降低AS斑块形成，调节血脂，稳定易损斑块，其机制与激活AMPKα1、AMPKα2及抑制STAT1表达以下调促炎因子 TNF-α、MMP-1、MMP-9、ICAM-1表达有关。本研究可拓展益气活血方药防治 AS 及其并发症的应用范围；本研究调控的信号蛋白有望成为临床防治AS的重要靶点。