Influence of red clover (Trifolium pratense) isoflavones on gene and protein expression profiles in liver of ovariectomized rats

Abstract 

Isoflavones such as genistein, biochanin A, formononetin, and glycetin are fairly abundant in red clover (Trifolium pratense, Fabaceae) and show estrogenic, antioxidant and hypolipidemic activities. To explore these effects mediated by red clover extract at the gene and protein levels, female ovariectomized rats were treated with an isoflavone rich extract of T. pratense. The experimental rats were divided into 2 groups of five animals each: a) control b) experimental group (red clover extract treated with 450mg/kg/day for four days). The treatment influenced the plasma lipid levels differentially. Plasma LDL concentrations were significantly reduced (p<0.05), whereas triglycerides increased (p<0.05). Plasma HDL and total cholesterol remained unchanged. The rat livers were examined for their differential gene expression by Affymetrix Rae230 DNA microarrays. In addition, the total liver proteins were separated by 2D PAGE and proteins, which showed differences in their intensities were identified by MALDI-TOF-MS. The extract influenced the transcript levels of many novel estrogen and non-estrogen responsive genes as well as other regulatory genes. Functional annotations indicate that genes involved in metabolic pathways, information processing, membrane transport regulation, signal transduction and other cellular processes were regulated. Quantitative reverse transcription analysis with real-time PCR confirmed that red clover extract regulates genes involved in lipid metabolism and antioxidation mechanisms. Proteomic analysis support the potential of red clover extract to modulate the lipid metabolism. In summary isoflavone rich red clover extract mediates numerous genomic and non-genomic effects, which influence besides the lipid metabolism a broad range of cellular functions, including metabolic actions, cell cycle regulation and antioxidant activity.

Keywords: DNA microarray, Gene expression profiling, Red clover, Lipid metabolism, Antioxidant mechanisms