新放线菌资源姜氏菌目菌株抗炎活性成分挖掘和作用机制研究

The excessive inflammation response results in persistent tissue damage，which plays a key role in the developments of arthritis, diabetes, arterial atherosclerosis and other diseases. New anti-inflammatory drugs discovery would be beneficial to inflammatory diseases treatment. In the course of our investigations on novel natural products from microbial sources for drug discovery, we found that the Jiangellales strains showed strong anti-inflammatory activity.. New Jiangellales is the highest taxonomic rank found by microbiologist in China. Because there is at least a 5% difference in the 16S rRNA gene sequence between the genera Jiangella and Haloactinopolyspora and other genera in Actinobacteria, the order Jiangellales could be considered as a novel microbial source for the discovery of novel metabolites. Currently, only six strains have been found from Jiangellales, including Jiangella gansuensia, J. alkaliphila, J. alba, J. muralis, Haloactinopolyspora alba and one similar strain to J. alba from animal faeces. Till now, no intensive reports concerning secondary metabolites generated by the bacterial strains in the order Jiangellales have been released except our group's work. We have evaluated the anti-inflammatory activity of the six strains fermentation broth extracts by bioassay. All the extracts exhibited inhibitory effects on lipopolysaccharide-induced NO production in RAW 264.7 macrophage cells, with 50~70% inhibition ratio at the dosage of 100 ug/mL. The preliminary investigation of the secondary metabolites have been performed on the strains. Five new anti-inflammatory active macrolactams were isolated and elucidated, which significantly reduced both the secretion of IL-1b, IL-6, TNF-a and the mRNA levels.. In order to explore new anti-inflammatory lead compounds, we will investigate the secondary metabolites on six Jiangellales strains systematically in this project. The bioactivities of the isolated compounds will be evaluated through anti-inflammatory bioassay in vivo and in vitro. Meanwhile, we will investigate the anti-inflammatory mechanism of new macrolactams found from the stains in the project. At the same time, We will clarify the specificity of taxonomy about the Jiangellales on the basis of secondary metabolites and provide the foundation for research and development of the Jiangellales.

炎症反应涉及多种疾病的病理过程，严重影响人类的健康水平，新型抗炎药物的发现对于炎症性疾病的治疗具有重要意义。课题组在从新放线菌资源寻找活性化合物过程中发现姜氏菌目菌株具有较好的抗炎活性。姜氏菌目是2012年新提出的分类单元，共发现6个菌株。除课题组前期工作外，该目菌株化学和生物活性研究工作未见报道。在对菌株次生代谢产物初步研究中得到5个大环内酰胺类新骨架化合物能够明显抑制Raw264.7细胞炎症因子IL-1b, IL-6和TNF-a表达水平。本课题拟对姜氏菌目6株菌株进行深入系统的化学成分研究，通过体内外活性评价对抗炎活性化合物进行深入挖掘；采用分子生物学手段对发现的新型大环内酰胺类抗炎成分进行深入作用机制探讨，明确化合物的作用靶点。拟通过本项目研究获得新型抗炎活性先导化合物；以代谢产物为起点，阐明该目菌株在分类学和遗传学方面的特异性，为姜氏菌资源的进一步开发和利用提供理论和实验基础。

本项目选择分类级别较高的一类全新的放线菌资源姜氏菌目菌株作为研究对象。对4株姜氏菌的代谢产物及代谢产物的抗炎活性进行了研究。同时在课题经费支持下对3株分离自动物粪便和地衣的链霉菌的代谢产物进行了研究。从7株菌的发酵液共得到109个代谢产物，包括24个新化合物。对大部分代谢物的抗炎活性及活性机制进行了研究。结果表明分离自姜氏菌的一类生物碱jiangrines，来自动物粪便菌的一类大环内酰胺macroviolactams和一类二氢色原酮类violacins化合物表现了良好的抗炎活性。对这几类化合物抗炎分子机制研究表明主要是通过MAPK和 NF-κB 信号通路来调节抑制炎性因子的产生。课题中对结构较简单的抗炎活性化合物violacin A 进行了全合成研究，并对合成的violacin A和violacin A衍生物抗炎活性进行了评价。姜氏菌目菌株作为一类分类级别较高的放线菌，具有代谢产生新化合物的能力。且同属不同菌种产生的代谢产物具有很大的相似性，代谢产物研究结果进一步从化学角度佐证分类学的正确性。从姜氏菌中发现的3个抗炎活性先导物jiangrines和violacin A均具有进一步研究和开发的价值。课题的研究对姜氏菌目菌株资源，化学与活性研究以及进一步作为抗炎药物先导物资源的开发和利用提供了实验基础和参考依据。