180 likes | 323 Views
Effect of a traditional Chinese herbal formula on estrogen receptor transactivation in HepG2 cells and serum cholesterol profile in hamsters. LK Leung Food & Nutritional Sciences/Biochemistry Chinese University of Hong Kong. Background.
E N D
Effect of a traditional Chinese herbal formula on estrogen receptor transactivation in HepG2 cells and serum cholesterol profile in hamsters LK Leung Food & Nutritional Sciences/Biochemistry Chinese University of Hong Kong
Background • Envied the market share enjoyed by a company who was selling a health product for relieving peri-menopausal syndromes. • A herb formula (GS) serving the same purposes was obtained from Shanghai. • JCICM was inviting proposals for developing TCM products.
Questions: • Was the herbal formula estrogenic? • What could be our testing assays/models?
ERa Competition Assay • Methodology: Recombinant ERa protein was coated on wells. Binding competition for ERa: Test compound competed with estradiol conjugated to a fluorescent probe. Fluorescence inversely proportional to binding affinity.
Estradiol labeled with a fluorescent probe Test compound ERalpha receptor
ERE-Luciferase Assay on HepG2 cells expressing ER Methodology: CMV driven ER expression vector ERE-Luciferase construct Lipofectamine
substrate Light Luciferase ER ERE driven luciferase reporter construct ER expression vector
ERE-Luciferase Assay on HepG2 cells expressing ER Methodology • Why HepG2 cells? 1 2 3 ERα 66kDa β-actin Lane1: HepG2 transfected with ER + one day recovery Lane2: HepG2 transfected with ER Lane3: HepG2 transfected with pcDNA3.1
Testing of Estrogenic Compounds by ERE-Luciferase Assay Po et al., Br J Nutr., 2002
Po et al., Cancer Letts, 2002 Testing of Anti-Estrogenic Compounds by ERE-Luciferase Assay
GS GS GS Effect of GS & Estrogen Co-treatment on ERa Transactivation in HepG2 Cells Effect of GS-Estrogen co-treatment on ERa transactivation
GS GS GS Effect of GS & Estrogen Co-treatment on ERb Transactivation in HepG2 Cells Effect of GS-Estrogen co-treatment on ERb transactivation
Findings in HepG2 Cells • GS is estrogenic and can bind to both ERa and ERb. • GS is not an antagonist to ERa, but somewhat suppresses the binding of E2 to ERb. What’s Next?
Cholesterol Study Using Hamster Model • High plasma cholesterol concentration than the other animal models (Lee et al., 1959). • Low hepatic synthesis of cholesterol and sensitive to dietary intake (Turley et al., 1995). • Resistant to the progression of atherosclerosis (Bauer, 1996).
Table 1. Effect of herbal extract (GS) administration on triglyceride, HDL-C and non-HDL-C in OVX hamsters. • OVX female (n=8-10) hamsters were treated with various concentrations of GS extract. Their serum triglyceride, HDL-C and non-HDL-C levels were determined after 4 weeks of treatment. Each value represents average ± SD of two independent experiments. *p<0.05 or **p<0.01 when compared with control.
Acknowledgements The Chinese University of Hong Kong Amanda Yee-man Yuen, Zhen-yu Chen, Jun Wang Shanghai Innovative Research CenterYong-zheng Hui, Quan-zhong Sun, Hong Kong Jockey Club Institute of Chinese Medicine Hong-xi Xu CIIT Centers for Health Research Ms. Susan Maness Duke University Dr. Donald McDonald This project is funded by the Hong Kong Jockey Club Charities Trust.