基于LOX-1/NF-κB/NLRP3信号轴探讨铁皮石斛叶黄酮类化合物抗动脉粥样硬化的药效物质基础及分子机制

IL-1β is an important cause of atherosclerosis and NLRP3 inflammasome is a key protein that mediates the expression of IL-1β. Previous studies have shown that flavonoids from Dendrobium officinaie leaves can lower blood lipid level and the expression of IL-1β, suggesting that it has the potential of anti-atherosclerotic effect. Based on previous study, in this project, we will establish ApoE-deficient mouse model of atherosclerosis and oxLDL-induced HAECs injury model firstly. Then, taking the inflammatory reaction as a breakthrough point, the effects of LOX-1/NF-κB/NLRP3 signal axis on prevention and treatment of atherosclerosis will be confirmed by studying the expression of LOX-1, NF-κB, NLRP3 and inflammation-related proteins using chemical inhibitors method and RNA interference technology. In additon, we will further reveal the substances basis and molecular mechanism of anti-atherosclerotic effects of flavonoids from Dendrobium officinaie leaves based on the LOX-1/NF-κB/NLRP3 signal axis. This study is from the anti-inflammatory point of view to enrich targets and small molecule compounds for the prevention and treatment of atherosclerosis, and it also provides a theoretical basis for the development of new drugs and healthcare products of Dendrobium officinaie leaves.

IL-1β是动脉粥样硬化发生的重要诱因，NLRP3炎性小体是介导IL-1β表达的关键蛋白。前期研究结果显示铁皮石斛叶黄酮类化合物能够降低血脂和降低IL-1β的表达，提示其具有潜在的抗动脉粥样硬化作用。本项目拟在此基础上，制备ApoE-/-小鼠动脉粥样硬化模型和oxLDL诱导HAECs损伤模型，以炎症反应为切入点，通过化学抑制剂和RNA干扰技术探索LOX-1、NF-κB、NLPR3以及炎症相关蛋白的变化，确证LOX-1/NF-κB/NLRP3信号轴在防治动脉粥样硬化的作用，并通过LOX-1/NF-κB/NLRP3信号轴揭示铁皮石斛叶黄酮类化合物抗动脉粥样硬化药效物质基础及分子机制。本研究从抗炎角度丰富了防治的靶点及其小分子化合物，为铁皮石斛叶新药及其保健品的开发提供理论依据。

以NLPR3为靶点的旨在调控炎症的药物开发是防治动脉粥样硬化的新策略。本项目利用LC-MS分析了铁皮石斛叶黄酮类化合物组成成分，并在氧化型低密度脂蛋白、脂多糖、双氧水诱导人主动脉内皮损伤模型筛选其活性成分，发现并确证抗炎的活性成分有：柚皮素；槲皮素；芹菜素；芦丁；异鼠李素；异夏佛塔苷；夏佛塔苷；异槲皮苷。然后进一步在细胞和/或整体水平上研究铁皮石斛叶黄酮类化合物对动脉粥样硬化的保护作用，动物实验结果显示紫杉叶素均能降低ApoE小鼠血清中TC、TG、LDL水平；升高HDL水平;减少主动脉斑块的形成；降低血清中IL-6、IL-1β、TNF-α的含量；降低主动脉组织中LOX-1、p-NF-κB、NLRP3、cleaved-caspase-1、cleaved-IL-1β等蛋白表达。在应用抑制剂和/或siRNA干扰的基础上，从细胞水平确认了铁皮石斛叶总黄酮或紫杉叶素能减轻氧化型低密度脂蛋白诱导人主动脉内皮凋亡，减少IL-6、IL-1β、TNF-α的含量；降低细胞中LOX-1、p-NF-κB、NLRP3、cleaved-caspase-1、cleaved-IL-1β等蛋白表达，从而最终在体内和体外水平全面确证了LOX-1/NF-κB/NLRP3信号轴在动脉粥样硬化发生、发展中的重要作用，以及铁皮石斛叶黄酮类化合物能够靶向调控其来防治动脉粥样硬化。此外，本研究首次表征了铁皮石斛叶多糖结构，其主要由甘露糖、葡萄糖、半乳糖、阿拉伯糖和半乳糖醛酸组成，进一步活性研究显示铁皮石斛叶多糖能够调控TLR4/NF-κB/NLRP3信号轴来阻止炎症的级联起到保护胃粘膜损伤和AMPK/mTOR/LC3β信号轴防治急性胃粘膜损伤的作用。综上，本项目的研究结果将对铁皮石斛叶新食品资源和保健品的开发具有指导作用。本项目已发表SCI论文4篇，另有2篇SCI论文待发表；辅助培养毕业硕士研究生2名，独立培养在读研究生1名。