| Cardiovascular disease is one of the most common diseases that damage human health in developed countries and most developing countries, while atherosclerosis is the principal pathogenesis for many critical cardiovascular diseases. Statins, a type of clinical routine drugs, reduce cardiovascular events by only about20%to40%. To lower the risk of cardiovascular disease and find drugs with novel mechanism of action, in addition to decrease low density lipoprotein cholesterol, a new preventative and/or therapeutic target for atherosclerosis need to be introduced. Recent studies revealed that the antiatherosclerotic effect of HDL was mainly attributed to its role in reverse cholesterol transport (RCT). Scavenger receptor class B type I (SR-BI) is the high-affinity HDL receptor at the molecular level, and the CLA-1(CD36and Lysosomal integral membrane protein-â…¡ Analogous-1) is the human HDL receptor. CLA-1/SR-BI has been suggested as a potential new preventative and/or therapeutic target for atherosclerosis due to its pivotal role in RCT. Promoting reverse cholesterol transport will decrease cholesterol in atherosclerotic plaques and reverse the developmental process of atherosclerosis. Thus increase of SR-BI/CLA-1expression will lead to the acceleration of RCT and the clearance of abundant cholesterol ester. As a result, the active compounds which enhance SR-BI/CLA-1expression may be developed as drug candidates or lead compounds for new antiatherogenic agents.Actinomycete strain04-9179was chosen for further isolation and characterization of active compounds. The fermentation filtrate was applied to HP-20macroporous resin column, which was washed with water and eluted with gradient acetone. The50%acetone eluate fractions enhancing luciferase activity were collected and concentrated in vacuo. The dryness was dissolved in a small methanol and subjected to a column of the octa decyltrichloro silane (ODS), and gradient eluted with three bed column of MeOH. Final purification was achieved using semipreparative HPLC. Three pure compounds9179B,9179D and9179E were isolated from fermentation broth of actinomycete strain04-9179. The structure of9179B,9179D and9179E was determined by means of spectral data of UV, ESI, ESI-MS,1H-NMR,13C-NMR,1H-1H COSY, DEPT, HSQC, NOSY and HMBC with CD3OD as the solvents. Finally,9179B,9179D and9179E were identified as (S,2E,4E)-7-(4-(dimethylamino)phenyl)-4,6-dimethyl-7-oxohepta-2,4-dienamide,(2E,4E,6S)-7-(4-(dimethylamino)phenyl)-4,6-d imethyl-7-oxo-N-((2R,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)hepta-2,4-dienamide and (S,2E,4E)-7-(4-(dimethylamino)phenyl)-N,4,6-trimethyl-7-oxohepta-2,4-dienamide. Positive compounds9179B,9179D and9179E significantly influenced CLA-1transcriptional activity at low micromolar concentrations in the developed assay. The up-regulatory activities were demonstrated at the transcriptional and protein levels in treated HepG2cells. Furthermore, possible effective sites of three active compounds9179B,9179D and9179E were identified by primary studies targeting PPARy and their influences on different cis elements in CLA-1upstream regulatory sequence.9179B,9179D and9179E could increase the uptake of Dil-labeled HDL by treated RAW264.7cells. In addition,9179B can influence ABCA1transcriptional activity and protein level of HepG2cells at low micromolar concentrations.This study identified for the first time that FL657C, Trichostiain D and Trichostatin RK have up-regulating activity on human high density lipoprotein receptor CLA-1and ATP-binding cassette transporter ABCA1, which bears important theoretical and practical significance for further investigation of the transcriptional regulation of the CLA-1/SR-BI gene and the discovery of drug candidates or lead compounds of new antiatherosclerotic agents. |
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