基于AMPK-PPARγ-LXRα通路介导的巨噬细胞胆固醇流出探讨调肝导浊方防治AS的机制

The high accumulation of cholesterol in macrophages induced to foam cells is an early pathological feature of atherosclerosis (AS), while the dysregulation of cholesterol efflux is the key factor lead to macrophages foaming and AS plaques forming. Researchs showed that cholesterol efflux in macrophages is mainly mediated by the surface receptor ABCA1 and ABCG1. If the receptor expression is decreased, the restriction of cholesterol outflow promotes the formation of foam cells. It was pointed out that PPAR gamma-LXR alpha pathway could increase the expression of ABCA1 and ABCG1 in macrophages to promote the macrophage cholesterol efflux and reduce the formation of AS plaques. Another study showed that AMPK may also enhance ABCA1 and ABCG1 expression in macrophages, which may be associated with the regulation of PPAR gamma-LXR alpha pathway. Traditional chinese medicine considered that AS belongs to the categories of stroke and chest discomfort. The main pathogenesis of AS is Qi stagnation in early stage, sputum accumulated with blood stasis in metaphase, and sputum combined with blood stasis in late stage. So the therapeutic Principle of AS must be relieving the depressed liver, regulating Qi activity and promoting coronary circulation in order to eliminate phlegm and descend turbid. The Tiaogan Daozhuo decotion created by Zhang Boli academician is effective for clinical prevention and treatment of AS, which could reduce plasma cholesterol levels, activate AMPK signal, and decrease the area of AS plaques in previous studies. Whether the mechanism of Tiaogan Daozhuo decotion correlation with the regulation of cholesterol efflux in macrophages or not has not been reported. Accordingly, our group proposes the hypothesis that Tiaogan Daozhuo decotion can promote macrophage cholesterol outflow and reduce the formation of AS plaques through activating AMPK-PPARγ-LXRα pathway to increase the expression of ABCA1 and ABCG1 in macrophages. Thus, we use ApoE knockout mice feeding with high-fat as AS model in vivo and RAW264.7 macrophages induced by ox-LDL as lipid-loaded model in vitro to analyze the relationship between AMPK-PPARγ-LXRα pathway and cholesterol efflux in macrophages, and ultimately illuminate the mechanisms of Tiaogan Daozhuo Decotion on the regulation of lipid metabolism and the prevention of AS.

巨噬细胞内胆固醇蓄积形成泡沫细胞是AS的早期病理特征，而胆固醇流出障碍是导致巨噬细胞泡沫化、促进AS斑块形成的关键。研究指出巨噬细胞内胆固醇主要通过ABCA1/ABCG1受体主动介导流出。PPARγ-LXRα信号可上调ABCA1/ABCG1的表达以减少AS斑块形成。相关研究发现AMPK亦可增强上述受体活性，其作用可能与调控PPARγ表达有关。调肝导浊方为张伯礼院士临床防治AS的经验有效方，前期研究证实其具有降脂、减少AS斑块的作用，然其机制是否与调控巨噬细胞胆固醇流出有关尚待阐明。据此，本课题组提出假说：调肝导浊方基于AMPK-PPARγ-LXRα通路上调巨噬细胞ABCA1/ABCG1表达，促进胆固醇流出以减少AS斑块形成。拟以高脂饲养ApoE-/-小鼠诱导AS模型、ox-LDL诱导巨噬细胞建立荷脂模型为研究对象，分析该通路与巨噬细胞胆固醇流出的相关性，阐明调肝导浊方防治AS的分子机制。

巨噬细胞内胆固醇蓄积形成泡沫细胞是AS的早期病理特征，而胆固醇流出障碍是导致巨噬细胞泡沫化、促进AS斑块形成的关键。调肝导浊方为张伯礼院士临床防治AS的经验有效方，前期研究证实其具有调脂、延缓AS进展的作用，然其深入机制尚待阐明。本项目组采用高脂饲养ApoE-/-小鼠12周复制AS模型，通过小动物超声、油红O染色、HE染色、Masson染色，明确调肝导浊方可减少AS斑块形成及斑块内脂质蓄积；再检测血脂水平发现调肝导浊方可能参与胆固醇代谢；最后通过qPCR及蛋白印迹发现调肝导浊方可上调AMPKα1、PPARγ、ABCA1、ABCG1的表达。离体通过50μg/ml ox-LDL诱导RAW264.7巨噬细胞24h复制泡沫细胞模型，通过油红O染色、游离胆固醇检测、胆固醇流出试验，明确调肝导浊方可促进巨噬细胞胆固醇流出；进而验证了调肝导浊方可上调巨噬细胞AMPKα1、PPARγ、ABCA1、ABCG1的表达。最后通过AMPK激动剂及抑制剂与PPARγ基因沉默干预，明确了调肝导浊方主要是通过激活AMPK/PPARγ信号以上调ABCA1的表达，促进胆固醇流出。本研究涉及的信号蛋白有望成为临床防治AS的重要靶点。