基于LXRα-SREBP1-ABCA1/G1信号通路的益气活血化痰方调脂抗动脉粥样硬化机制研究

Reverse Cholesterol Transportation(RCT) against atherosclerosis(AS) is an important mechanism, Cholesterol efflux from macrophages is a critical step of RCT, ATP-Binding Casstte transportors A1/G1(ABCA1/G1) play a key role in mediating RCT, clinical studies demonstrated that Yiqi Huoxue Huatan Recipe(YHHR) could decrease serum lipid in patients with coronary heart disease, animal model studies show that the mechanism of YHHR anti AS is related to the upregulation of vascular Liver X Receptor alpha(LXRα), Sterol Regulatory Element Binding Protein1（SREBP1） and ABCA1/G1. According to this, we put forward a hypothesis: YHHR promotes RCT, anti AS, by activating the LXR α -SREBP1-ABCA1/G1 pathway of vascular intimal macrophages.This study intends to carry out: YHHR intervention AS model mice, the separation of vascular macrophages, detection of LXRα, SREBP1 and ABCA1/G1 expression; To establish the AS model of THP-1 macrophages in vitro, To block the signal of LXR α or SREBP1, respectively, and to detect cholesterol efflux rate and expression of ABCA1/G1;YHHR drug serum was used to interfere with AS in vitro, to study the regulation of cholesterol efflux rate and LXRα -SREBP1-ABCA1/G1 pathway, In order to clarify some of the modern molecular mechanism of promoting circulation of Qi and blood and dispersing phlegm method in the treatment of AS.

胆固醇逆转运（RCT）是抗动脉粥样硬化（AS）的重要机制，巨噬细胞胆固醇外流是RCT的关键步骤，ABCA1/G1在介导RCT中发挥关键作用。临床研究证实益气活血化痰方（YHHR）对冠心病患者具有降脂作用，动物模型研究发现YHHR抗AS的机制与血管LXRα、SREBP1和ABCA1/G1上调有关，据此提出假说：YHHR通过活化血管内膜巨噬细胞LXRα-SREBP1-ABCA1/G1通路，促进RCT，抗AS。本研究拟开展：YHHR干预AS模型小鼠，检测血管内膜巨噬细胞LXRα、SREBP1和ABCA1/G1的表达；建立THP-1巨噬细胞的AS体外模型，分别阻断LXRα或SREBP1信号，检测ABCA1/G1的表达和胆固醇流出率；YHHR药物血清干预AS体外模型，研究其对LXRα-SREBP1-ABCA1/G1通路的调节作用及胆固醇流出率，以期部分阐明益气活血化痰法治疗AS的现代分子机制。

背景：动脉粥样硬化性心血管疾病是最重要的高发疾病和主要死亡原因，居我国城乡居民总死亡原因的首位，是当前亟待解决的重大公共健康问题。脂质平衡功能失调是 ASCVD 发生发展的关键病理基础。益气活血化痰方的前期临床结果表明该方在治疗ASCVD上具有显著的疗效，但作用机制至今尚不明确。基因芯片结果提示益气活血化痰方作用机制可能与胆固醇转运相关。.目的：本研究探索LXRα-SREBP1-ABCA1/G1表达和胆固醇流出的关系，探讨益气活血化痰方抗AS的作用机制。.方法：在体实验采用 apoE-/-小鼠加高脂喂养致 AS 模型，进行药物干预后检测血清中脂质代谢指标；油红O染色半定量分析胸主动脉血管巨噬细胞内脂质沉积；WB和qPCR检测组织中LXRα、SREBP1和ABCA1/G1表达水平。体外实验以THP-1巨噬细胞为研究对象，以ox-LDL诱导促进胆固醇细胞内聚集，分别阻断或过表达LXRα或SREBP1信号，采用液体闪烁法测定放射性物质进出细胞，计算胆固醇流出率；高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量；WB和qPCR检测细胞中LXRα、SREBP1、ABCA1和ABCG1蛋白含量及其mRNA基因表达。.结果：LXRα可以靶向调控SREBP1；阻断LXRα后ABCA1/G1表达下降，巨噬细胞胆固醇流出减少；阻断SREBP1后ABCA1表达上调，胆固醇逆转运进一步增强，胞内胆固醇含量降低。益气活血化痰方减少主动脉壁厚度和动脉壁胶原纤维，降低脂质蓄积、血清总胆固醇含量和低密度脂蛋白胆固醇含量。促进LXRα、SREBP1、ABCA1/G1 蛋白和mRNA表达，对于SREBP1的过度表达有一定的拮抗作用，能够进一步影响ABCA1的转录，加快巨噬细胞源性泡沫细胞的胆固醇流出。.结论： LXRα-SREBP1-ABCG1信号途径存在正向调控关系，益气活血化痰方通过调控LXRα-SREBP1-ABCG1信号途径，靶向激动LXRα，上调ABCA1/G1表达，增强胆固醇逆转运，发挥抗AS或延缓AS发展的作用。