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

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EC₅₀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。

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

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 semipreparative 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 RTPCR 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 EC₅₀ value of 46.02 umol/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 Dil-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.