DataSheet1_Network pharmacology and experimental analysis to reveal the mechanism of Dan-Shen-Yin against endothelial to mesenchymal transition in atherosclerosis.ZIP

Atherosclerosis is a chronic inflammatory disease characterized by the formation of plaque and endothelial dysfunction. Under pro-inflammatory conditions, endothelial cells adopt a mesenchymal phenotype by a process called endothelial-to-mesenchymal transition (EndMT) which plays an important role in the pathogenesis of atherosclerosis. Dan-Shen-Yin (DSY) is a well-known traditional Chinese medicine used in the treatment of cardiovascular disease. However, the molecular mechanism whereby DSY mitigates atherosclerosis remains unknown. Therefore, we employed a network pharmacology-based strategy in this study to determine the therapeutic targets of DSY, and in vitro experiments to understand the molecular pharmacology mechanism. The targets of the active ingredients of DSY related to EndMT and atherosclerosis were obtained and used to construct a protein-protein interaction (PPI) network followed by network topology and functional enrichment analysis. Network pharmacology analysis revealed that the PI3K/AKT pathway was the principal signaling pathway of DSY against EndMT in atherosclerosis. Molecular docking simulations indicated strong binding capabilities of DSY’s bioactive ingredients toward PI3K/AKT pathway molecules. Experimentally, DSY could efficiently modify expression of signature EndMT genes and decrease expression of PI3K/AKT pathway signals including integrin αV, integrin β1, PI3K, and AKT1 in TGF-β2-treated HUVECs. LASP1, which is upstream of the PI3K/AKT pathway, had strong binding affinity to the majority of DSY’s bioactive ingredients, was induced by EndMT-promoting stimuli involving IL-1β, TGF-β2, and hypoxia, and was downregulated by DSY. Knock-down of LASP1 attenuated the expression of integrin αV, integrin β1, PI3K, AKT1 and EndMT-related genes induced by TGF-β2, and minimized the effect of DSY. Thus, our study showed that DSY potentially exerted anti-EndMT activity through the LASP1/PI3K/AKT pathway, providing a possible new therapeutic intervention for atherosclerosis.

动脉粥样硬化（Atherosclerosis）是一种以斑块形成与内皮功能障碍为特征的慢性炎症性疾病。在促炎条件下，内皮细胞可通过名为内皮细胞向间充质转化（endothelial-to-mesenchymal transition, EndMT）的过程获得间充质表型，该过程在动脉粥样硬化的发病机制中发挥关键作用。丹参饮（Dan-Shen-Yin, DSY）是一类常用于治疗心血管疾病的经典中药，但其缓解动脉粥样硬化的分子机制尚未阐明。因此，本研究采用基于网络药理学的策略以明确DSY的治疗靶点，并通过体外实验阐释其分子药理机制。本研究获取了与EndMT及动脉粥样硬化相关的DSY活性成分靶点，以此构建蛋白质相互作用（protein-protein interaction, PPI）网络，随后开展网络拓扑分析与功能富集分析。网络药理学分析显示，PI3K/AKT信号通路是DSY拮抗动脉粥样硬化中EndMT的核心信号通路。分子对接模拟结果表明，DSY的生物活性成分可与PI3K/AKT通路分子形成较强结合。实验结果显示，在经转化生长因子β2（TGF-β2）处理的人脐静脉内皮细胞（HUVECs）中，DSY可有效调控标志性EndMT基因的表达，并降低整合素αV、整合素β1、PI3K及AKT1等PI3K/AKT通路信号分子的表达水平。LASP1作为PI3K/AKT通路的上游分子，可与多数DSY生物活性成分具有较强结合亲和力；其可被EndMT促进刺激物（包括白细胞介素1β、TGF-β2及缺氧）诱导上调，且可被DSY下调。敲低LASP1可减弱TGF-β2诱导的整合素αV、整合素β1、PI3K、AKT1及EndMT相关基因的表达，并削弱DSY的干预效应。综上，本研究表明DSY可通过LASP1/PI3K/AKT通路发挥抗EndMT活性，为动脉粥样硬化的治疗提供了潜在的全新干预策略。