基于丹参酮小分子探针研究其抗血管内皮细胞氧化应激损伤的分子靶标及调控机制

Nuclear-factor-E2 related factor 2/antioxidant responsible element plays a crucial role in protecting vascular endothelial cells. In our previous study, twenty Nrf2 activators belong to tanshinones were screened from Danshen by high-resolution peak fractionation approach coupled with an ARE-luciferase reporter gene assay. Then we found the bioactive tanshinones protected vascular endothelial cells against oxidative stress-induced injury via the Nrf2-ARE pathway. But the mechanism of action of tanshinones in activating Nrf2 is still unknown. In the study, small molecular probes will be rationally designed according to the structure-activity relationship of tanshinones. Using the probes and affinity magnetic beads, the binding proteins will be captured and identified as potential targets by label-free quantitative proteomics techniques. Then the candidate targets will be validated by using multiple molecular biology methods. Based on the target, the regulation mechanism of tanshinones protecting vascular endothelial cells against injury could be illustrated. The study could not only reveal the mechanism of action of tanshinones, but also provide new scientific basis for using Danshen to treat atherosclerosis. It also provides a useful inspiration for the study of bioactive components and molecular mechanism of other TCM.

Nrf2-ARE信号通路在血管内皮保护中起着重要作用。我们前期应用高纯度馏分制备结合报告基因细胞筛选，从丹参中发现了20个丹参酮类Nrf2激活剂，进一步研究表明丹参酮活性成分可诱导Nrf2-ARE通路下游多种保护性基因的表达，发挥抗内皮细胞氧化应激损伤的作用，但此类成分激活Nrf2的分子机制尚不清楚。本项目基于丹参酮活性成分的构效关系，进行小分子探针的合理设计，以小分子探针为工具，应用亲和磁分离技术捕获结合蛋白，采用非标记定量蛋白质组学方法识别候选靶标，并运用多种分子生物学手段进行靶标验证，进而基于靶标从源头上阐明丹参酮类活性成分抗血管内皮损伤的调控机制。本研究不仅在分子水平阐明丹参酮活性成分的作用原理，而且为丹参抗动脉粥样硬化作用提供新的科学依据，也为其它中药活性成分研究和作用机制研究提供借鉴。

Nrf2-ARE信号通路在血管内皮保护中起着重要作用。本项目结合高纯度馏分制备-筛选-鉴定一体化方法，新建立了一种适于中药复杂体系的微量活性成分发现方法，以内皮细胞保护活性为导向，从丹参提取物中发现了38个具有激活Nrf2/ARE信号通路的二萜醌类成分，其中18个微量成分的活性为首次报道。针对发现的Nrf2激活成分丹参新酮、丹参二醇C、丹参酮IIA等化合物开展了深入的作用机制研究。初步明确了丹参酮类活性成分激活Nrf2-ARE通路的构效关系，以丹参酮IIA为前体化合物，共合成了12个新的具有血管内皮细胞保护作用的衍生物。进而设计并合成了2个生物素化丹参酮衍生物，以小分子探针为工具，应用亲和磁分离技术捕获结合蛋白，采用非标记定量蛋白质组学方法识别候选靶标并进行靶标验证。本研究不仅在分子水平进一步揭示了丹参酮活性成分调控Nrf2-ARE信号通路的新作用机制，并为丹参酮活性成分在与氧化应激发病机制相关疾病中的研究开发提供了新的科学依据。