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LK Leung Food & Nutritional Sciences/Biochemistry Chinese University of Hong Kong

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.

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LK Leung Food & Nutritional Sciences/Biochemistry Chinese University of Hong Kong

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  1. 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

  2. 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.

  3. Questions: • Was the herbal formula estrogenic? • What could be our testing assays/models?

  4. 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.

  5. Estradiol labeled with a fluorescent probe Test compound ERalpha receptor

  6. ERE-Luciferase Assay on HepG2 cells expressing ER Methodology: CMV driven ER expression vector ERE-Luciferase construct Lipofectamine

  7. substrate Light Luciferase ER ERE driven luciferase reporter construct ER expression vector

  8. 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

  9. Testing of Estrogenic Compounds by ERE-Luciferase Assay Po et al., Br J Nutr., 2002

  10. Po et al., Cancer Letts, 2002 Testing of Anti-Estrogenic Compounds by ERE-Luciferase Assay

  11. Effect of GS on ERa Transactivation in HepG2 Cells GS GS

  12. Effect of GS on ERb Transactivation in HepG2 Cells GS GS

  13. GS GS GS Effect of GS & Estrogen Co-treatment on ERa Transactivation in HepG2 Cells Effect of GS-Estrogen co-treatment on ERa transactivation

  14. GS GS GS Effect of GS & Estrogen Co-treatment on ERb Transactivation in HepG2 Cells Effect of GS-Estrogen co-treatment on ERb transactivation

  15. 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?

  16. 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).

  17. 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.

  18. 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.

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