三株南海放线菌中作用于PPAR-LXR-ABCA1通路的抗动脉粥样硬化活性次生代谢产物的发现

Atherosclerosis is the critical pathophysiological basis of cardiovascular disease such as myocardiol infarction, stroke, and so on. Presently, there is no effective treatment for such diseases. Since lipid metabolism disorder and inflammation are critical procedures during the course of atherosclerosis progression, it is important to discover anti-atherosclerosis compounds with anti-inflammatory and anti-lipid bioactivities from the natural sources. Previously, 89 marine actinomycetes were isolated from the sediment of the South China Sea. They were subjected to a small-scale fermentation using three different culture media of Y1, Y5, and Y6 to give 267 crude extracts. All these crude extracts were tested for anti-inflammatory and anti-lipid effect. As a result, three strains showed potent anti-inflammatory effect against LPS-induced TNFα and IL6 production. Moreover, they also exhibited significant anti-lipid effect by reducing the production of foam cells. Further investigation indicated that they might exert the effect through the classical lipid metabolism pathway of PPAR-LXR-ABCA1. On the basis of the above results, we plan to focus on these three actinomycetes to find out the anti-atherosclerosis secondary metabolites. Firstly, a large scale (20 L) fermenation will be conducted for the three actinomycetes. By MPLC-ELISA techniques, the bioactive secondary metabolites can be isolated very quickly from the fermentation broth. Finally, the mechanisms of the bioactive compounds will be investigated to orientate the action pathway and the specific targets. In general, through the investigation of this project, the anti-atherosclerosis lead compounds with anti-inflammatory and anti-lipid activities could be discovered from marine actinomycetes.

动脉粥样硬化（Atherosclerosis，AS）是心肌梗塞、中风等致死性心、脑血管疾病的主要病理生理基础。目前临床上对AS的治疗尚不尽人意。脂代谢紊乱和炎症的发生贯穿了AS的发生发展。因此，从具有抗炎降脂活性的天然产物中寻找抗AS药物是目前该类药物研发的一个重要方向。前期，我们对从我国南海海洋分离得到的89株放线菌进行了活性筛选，发现三株放线菌不仅具有抑制LPS诱导的TNFα和IL6生成的作用，而且还能显著地减少吞噬脂质后形成的泡沫细胞的数量。进一步的研究表明，他们可能是通过影响PPAR-LXR-ABCA1的通路起到了抗AS的作用。因此，本项目拟以该三株放线菌为研究对象，从中寻找发现抗AS的活性次生代谢产物，并确定活性成分的作用机理和具体的作用靶点。通过本项目的研究，有望从海洋放线菌中寻找到降脂不伤肝的新型抗动脉粥样硬化先导化合物。

海洋微生物是新颖结构和独特活性化合物的重要来源，是天然产物研究新的重点领域。本项目旨在前期研究的基础上，基于从具有抗炎降脂活性的天然产物中寻找抗动脉粥样硬化（Atherosclerosis，AS）药物的思路，以能显著减少吞噬脂质后形成的泡沫细胞数量的三株放线菌为研究对象，通过微生物发酵、代谢物提取、化合物分离、结构鉴定、活性测试等工作，发现其中的活性成分单体。项目执行三年来，我们通过对深海放线菌Williamsia sp. MCCC 1A11233、Nesterenkonia flava MCCC 1K00610、Microbacterium sp. MCCC 1A11207、Saccharopolyspora cebuensis MCCC 1A09850和深海真菌Botryotinia fuckeliana MCCC 3A00494进行系统的化学成分研究，从中一共分离了150个化合物，其中包括81个新化合物。我们对分离获得的全部化合物进行了抗AS活性筛选，并且从中发现了两个活性最好的化合物，aphidicolin A10和aphidicolin A43。MTT法显示，这两个化合物对RAW264.7细胞有抑制活性，IC50值分别为6.33和7.33 μg/ml；它们还显示对LPS刺激的RAW264.7产生的主要炎性细胞因子TNFα的抑制活性；它们能减少泡沫细胞的数目从而具有潜在降低血脂作用；并且它们均激活了PPAR-LXR-ABCA1通路。为了拓宽活性，我们还对已鉴定的次生代谢产物分别进行了抗菌、抗炎、抗肿瘤、抗过敏等活性的测试，发现深海放线菌Nesterenkonia flava MCCC 1K00610中分离得到的化合物cyclo(D)-Pro-(D)-Leu和indol-3-carbaldehyde显示出抗过敏活性，IC50值分别为69.95和57.12 µg / mL；放线菌Saccharopolyspora cebuensis MCCC 1A09850中分离得到的化合物cebulactam A2对Hela和H1299肿瘤细胞株具有一定的抗增殖活性。本项目的实施为从深海微生物来源的抗AS药物的研发提供了模式结构和药物前体。同时本项目筛选发现的若干其它活性物质也具有一定的研究价值，有望成为抗过敏、抗肿瘤药物先导化合物。