人高密度脂蛋白受体CLA-1上调剂9179D的分离、结构鉴定和活性研究  

Study of isolation,identification and biological activity of human high density lipoprotein receptor CLA-1 up-regulator 9179D

作　　者：徐扬[1] 许艳妮[1] 巫晔翔[1] 余利岩[1] 洪斌[1] 司书毅[1] 

机构地区：[1]中国医学科学院医药生物技术研究所,北京100050

出　　处：《中国抗生素杂志》2012年第5期377-382,共6页Chinese Journal of Antibiotics

基　　金：国家自然科学基金(No.81102443);十一五"重大新药创制"科技重大专项(No.2010ZX09401-403)

摘　　要：目的从微生物代谢产物中分离能够上调人高密度脂蛋白受体(CLA-1)表达的新型活性化合物,并对其活性进行研究。方法应用已建立的CLA-1表达上调剂的模型,对阳性菌株链霉菌104A-9179发酵产物的活性成分进行分离纯化,获得活性化合物9179D;通过理化性质、质谱、紫外和核磁等波谱学数据进行结构鉴定;利用RT-PCR和Western Blot方法检测9179D对HepG2中CLA-1表达的影响;利用流式细胞仪检测其对小鼠巨噬细胞RAW264.7结合DiI-HDL的影响。结果从链霉菌104A-9179发酵产物中得到活性化合物9179D,并确定了其结构为曲占柳菌素D(Trichostatin D);9179D在CLA-1上调剂模型上的EC_(50)为46.02μmol/L,表达活性最高值为934%;9179D能增加HepG2中CLA-1的mRNA和蛋白表达;增加RAW264.7对DiI-HDL的结合。结论得到一个微生物来源的具有强的上调CLA-1的表达活性的化合物-9179D(曲古柳菌素D),属首次报道。Objective To find new compound which can up-regulate human high density lipoprotein receptor CLA-1 expression from microbial secondary metabolites, and then study its biological activity. Methods By using a cell-based high throughput screening model for the CLA-1 up-regulator, Streptomyces I04A-9179 was found to produce up-regulators of CLA-1. By column chromatography of HP20 macroporous resin, ODS column and semi- preparative HPLC, an active compound, 9179D, was isolated from the broth of Streptomycetaceae I04A-9179. By means of EI-MS, ESI-MS, ^1H, and ^13C NMR spectra, the structure of 9179D was identified. Western blot and RT- PCR assays were used to evaluate the effect of 9179D on CLA-1 expression in HepG2 cells. DiI-HDL uptake assay was performed in RAW264.7 cells by flow cytometry. Results Compound 9179D identified as Trichostatin D was isolated and purified from the fermentation products of a positive strain I04A-9179. The maximal CLA-1 upregulatory activity of 9179D in a cell-based reporter assay reached to 934%, with an ECs0 value of 46.02μmol/L. 9179D also up-regulated CLA-1 expression at both mRNA and protein levels in HepG2 cells. And 9179D could also increase the uptake of DiI-HDL in RAW264.7 cells. Conclusion 9179D obtained from Streptomyces I04A-9179 was identified as Trichostatin D, and it is first reported as a potential small-molecular upregulator of CLA-1.

关 键 词：人高密度脂蛋白受体 上调剂 高通量筛选 微生物次级代谢产物 

分 类 号：R972.6[医药卫生—药品]