基于活性和液质联用双重导向的沉香中抗神经炎症药效物质和作用机制研究

Neurodegenerative diseases (ND) have been the major diseases which are the severe threat to human health. Previous studies have disclosed that neuroinflammation which is initiated by the activation of microglial cells in the brain acts significant roles from appearance to development of ND, suggesting it is a reasonable way to treat ND by suppressing the activation of microglial cells. Our previous study has demonstrated that the EtOAc extract of agarwood (Lignum Aquilariae Resinatum) and its characteristic constituents, 2-(2-phenylethyl)chromones, could significantly inhibit the LPS-induced activation of microglial cells, thus showing great potency for the treatment of ND. The objective of the proposed research is to discover, characterize, and validate the anti-neuroinflammatory components and their action mechanisms of agarwood. Specific Aim 1 will develop an activity and LC/MS dual-guided screening strategy based on LCMS-IT-TOF and LPS-activated BV-2 microglial cell model for mining active compounds from agarwood. Active compounds and their analogues will be purified through LCMS-IT-TOF-guided separation procedure, and their structures are elucidated using combinatory spectroscopic and spectrometric methods. Specific Aim 2 will evaluate the anti-neuroinflammatory effects of the isolated compounds against LPS-activated BV-2 microglial cells. Quantitative structure-activity relationship (QSAR) study will be performed and the essential substructural units for the activities will be revealed. The anti-neuroinflammatory effects of active compounds will be further validated in vivo through animal models. Specific Aim 3 will characterize anti-neuroinflammatory effects of active compounds at the molecular and cellular levels, and disclose the signaling pathways and targets being responsible for their anti-neuroinflammatory activities. Accomplishing these objectives will afford new insights into the clinical use of agarwood and its prescriptions, and also provide drug leads for neuroinflammation-related diseases.

神经退行性疾病已成为严重威胁人类健康的重大疾病，脑内小胶质细胞活化介导的神经炎症贯穿该类疾病的发生、发展全过程，抑制小胶质细胞活化成为防治此类疾病的重要策略。沉香为名贵中药材，前期研究发现沉香乙酸乙酯提取物能够明显抑制LPS诱导的小胶质细胞活化，并获得4个活性较好的2-(2-苯乙基)色酮类化合物。本课题拟以LPS诱导的小胶质细胞活化模型联合LC/MS-IT-TOF准确识别为技术依托，建立“基于活性和液质联用双重导向的中药药效物质研究体系”，对沉香中抗神经炎症的活性成分及其结构类似物进行快速识别和高效制备；通过进一步活性验证，阐明其活性成分，并探讨其构效关系；对强活性成分进行体内活性验证，并从细胞、分子水平阐明其作用靶点和信号通路，为沉香及其复方临床用于神经退行性疾病的防治提供理论基础和科学依据，并为防治神经退行性疾病新型药物的发现提供先导分子，具有重要的学术价值和应用前景。

神经退行性疾病已成为严重威胁人类健康的重大疾病。脑内小胶质细胞活化介导的神经炎症贯穿该类疾病的发生、发展全过程，抑制小胶质细胞活化成为防治此类疾病的重要策略。沉香为名贵中药，课题组前期研究发现沉香乙酸乙酯提取物能够明显抑制LPS诱导的小胶质细胞活化，并分离得到4个活性较好的2-(2-苯乙基)色酮类化合物。本项目以抑制BV-2小胶质细胞活化活性为导向，结合LC-MS/MS液质联用技术对沉香95%乙醇提取物的石油醚和乙酸乙酯活性部位进行了系统的导向性化学成分研究，共分离并鉴定了73个化合物，其中新化合物39个。具体包括26个倍半萜类化合物、11个简单2-(2-苯乙基)色酮类、4个四氢2-(2-苯乙基)色酮类、30个2-(2-苯乙基)色酮二聚体以及2个其他类化合物。通过脂多糖（LPS）诱导BV-2小胶质细胞释放一氧化氮（NO）模型进行体外活性筛选。结果发现5个倍半萜类单体化合物能不同程度地抑制脂多糖诱导的BV-2小胶质细胞NO的释放，显示出潜在的抗炎活性。对苯乙基色酮类化合物GYF-21进行深入活性研究，我们发现该化合物能够显著抑制LPS刺激的巨噬细胞NO的合成、可以显著抑制小胶质细胞、树突状细胞及中性粒细胞等天然免疫细胞的活化、对在Th17细胞分化中具有较为重要作用的IL-6、IL-1β、IL-23等细胞因子分泌有显著的抑制、能够抑制CD4+T细胞的分化、可以抑制CD8+T细胞的增殖和IFN-γ的释放，表现出了良好的天然免疫和适应性免疫抑制作用，具有成为新型小分子免疫抑制剂的巨大潜力。发表论文14篇，其中SCI论文6篇，核心期刊4篇。申请专利2项。培养青年科研骨干3名，硕士研究生3名。