基于中性粒细胞弹性蛋白酶(NE)抑制的民间药物白菊木中新颖的抗炎萜类成分的发现及其作用机制研究

Neutrophil elastase (NE), as one of the important target protein, play a crucial role in regulation of inflammation, especially lung inflammation. So far, only one registered chemically synthesized selective NE inhibitor, named sivelestat, was applied to clinical treatment. It has great potential on research and development of NE inhibitor (NEI) from natural medicine. Gochnatia decora (Kurz) A. L. Cabrera, is a rare woody plant in Compositae. Its bark has been used for the treatment of pneumonia, cough, and wound in the areas of Lincang and Xishuangbana in China, but there is no reports about its active substance and pharmacological action. In our previous study, many diterpenes and triterpenes were isolated and identified from this plant, some of which displayed obvious inhibitory effect on NE and antiinflammatory activity in vitro. It suggested that those terpenoids are the main active material with efficacy of clearing lung and relieving cough in G. decora, and their action mechanism is related to NE inhibition. Based on the above research, this project will continue to isolate more novel terpenoid molecules from G. decora by the guide of NE inhibitory bioassays and UPLC-DAD-Q-TOF-MSn analysis, clarify antiinflammatory material basis of G. decora by the pharmacological experiments in vivo and vitro, and elucidate the mechanism related NE inhibition of active terpenoids using immunohistochemical and proteomic methods, such as RT-PCR and Western boltting, etc. This study focused on discovery of more novel NEI from plant resources and clarification of their action mechanism. It provides fundamental research on the development of more lead compounds with NE inhibition from natural medicine.

中性粒细胞弹性蛋白酶(NE)是参与调控炎性(特别是肺部炎症)反应的重要靶标蛋白之一，迄今只有一个作用于该靶蛋白的合成药物西维来司钠进入临床，开发前景广阔。白菊木为我国临沧、版纳地区民间常用药，主治肺热喘咳，至今尚无药效物质及药理研究报道。申请人前期从白菊木中发现一系列具有良好NE抑制及抗炎活性的萜类成分，可能为其清肺止咳之功的药效物质。本项目拟在此基础上，以NE抑制活性为导向，运用UPLC-Q-TOF-MSn技术，继续定向分离白菊木中结构新颖的萜类成分，结合体内外活性实验，阐明白菊木清肺止咳药效物质基础。运用免疫组化、RT-PCR，Western blotting 等方法，研究具有NE抑制活性的分子对NF-κB信号通路的影响，进一步阐明白菊木药效物质作用机制。本项目致力于从丰富的植物资源宝库中研究发现结构新颖的NE抑制剂并阐明其作用机制，为开发更多天然NE蛋白酶靶向先导药物提供前期研究。

白菊木是一种罕见的菊科木本植物，其树皮在我国滇南地区具有民间药用历史，主要用于治疗肺热喘咳、枪伤刀伤等，至今尚无药效物质及药理研究报道。本项目运用现代化学分离方法及UPLC-Q-TOF-MSn定向检测技术，对白菊木树皮及其枝叶部分进行了系统的化学成分研究；以其传统功效为依据，对白菊木化学成分进行了包括抑制中性粒细胞弹性蛋白酶(NE)等一系列抗炎活性研究。项目截至目前，已从白菊木树皮及其枝叶甲醇提取物中分离并鉴定单体化合物58个，其中包含全新骨架的二萜类化合物2个，新颖的对映-贝壳衫烷型二萜及其苷类化合物16个，新颖的倍半萜类化合物4个。药理研究显示，白菊木树皮甲醇提取物 (10 μg/mL) 具有一定的NE抑制活性 (抑制率>40 %)，树皮及枝叶提取物均可不同程度的抑制LPS诱导的小鼠巨噬细胞RAW264.7产生的NO，ROS，TNF-α，IL-6等炎症因子，对LPS诱发的急性肺炎小鼠肺部炎性细胞浸润具有一定的抑制效果；同时通过以上细胞模型，分别发现12个萜类单体化合物具有一定的NE抑制活性 (100 μM，抑制率>40 %) 及良好的一氧化氮 (NO) 抑制活性 (IC50<10 μM)。以上结果提示我们，白菊木中的含有大量的萜类化合物，且具有一定的抗炎活性，可作为白菊木发挥其治疗肺部炎作用的相关物质基础进行更加深入的研究。