Molecular mechanisms underlying the cholesterol-lowering effect of Ginkgo biloba extract in hepatocytes: a comparative study with lovastatin

Abstract

Aim: To explore the molecular mechanisms underlying the cholesterol-lowering effect of a Ginkgo biloba extract (GBE). Methods: Enzyme activity, cholesterol flux and changes in gene expression levels were assessed in cultured hepatocytes treated with GBE or lovastatin. Results: GBE decreased the total cholesterol content in cultured hepatocytes and inhibited the activity of HMG-CoA reductase, as determined by an in vitro enzyme activity assay. In addition, GBE decreased cholesterol influx, whereas lovastatin increased cholesterol influx. GBE treatment induced significant increases in the expression of cholesterogenic genes and genes involved in cholesterol metabolism, such as SREBF2, as determined by cDNA microarray and real-time RT-PCR. Furthermore, INSIG2, LDLR, LRP1, and LRP10 were differentially regulated by GBE and lovastatin. The data imply that the two compounds modulate cholesterol metabolism through distinct mechanisms. Conclusion: By using a gene expression profiling approach, we were able to broaden the understanding of the molecular mechanisms by which GBE lowers cellular cholesterol levels. Specifically, we demonstrated that GBE exhibited dual effects on the cellular cholesterol pool by modulating both HMG-CoA reductase activity and inhibiting cholesterol influx.Keywords: Ginkgo biloba extract; cholesterol; HMG-CoA reductase; influx; lovastatin; microarray Acta Pharmacologica Sinica (2009Sinica ( ) 30: 1262Sinica ( -1275 doi: 10.1038/aps.2009 published online 24 August 2009 Original ArticleThe original microarray data have been submitted to GEO (accession number GSE11830). * To whom correspondence should be addressed. E-mail qinghua_zhang@shbiochip.com cholesterol-lowering effect of GBE, we used a combination of experimental approaches, including enzyme activity and cholesterol flux assays, along with cDNA microarray and real time RT-PCR, to explore the gene regulation networks that are downstream of GBE treatment. Data presented here bring a new understanding to the molecular mechanisms by which GBE affects cholesterol metabolism. Materials and methodsCell culture and treatment GBE was provided by XingLing Pharmaceutical Co (Shanghai, China) as a standardized product that contains ~24% flavonol glycosides, ~6% terpene lactones (ginkgolides, bilobalide), and less than ~5 ppm ginkgolic acid. HepG2 cells were maintained in Minimum Essential Medium (MEM, Invitrogen) with 10% fetal bovine serum (FBS) (JRH Biosciences). Cells were seeded in 6-well plates at a density of 4×10 5 cells per well. When the cells were about 70% confluent, the medium was replaced with medium containing 2% FBS for drug treatment experiments. For dose-response experiments, a variety of concentrations of GBE (50, 100, and 200 µg/mL) were used, and the cells were treated for 24 h. For time course experiments, cells were treated for different times (6,12,24, and 48 h) with either 200 µg/mL of GBE or 0.5 μmol/L lovastatin. Control cells were treated with 0.1% DMSO (the vehicle) for the longest treatment time in the experiment. Tre...

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