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Analysis of the interaction of garlic with mediators of inflammation involved in the molecular mechanism of atheroscle

Workpackage N o 5.1. Analysis of the interaction of garlic with mediators of inflammation involved in the molecular mechanism of atherosclerosis. Year 1 and 2 . Influence of „garlic“ on the activation of the transcription factor NF-kappaB

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Analysis of the interaction of garlic with mediators of inflammation involved in the molecular mechanism of atheroscle

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  1. Workpackage No 5.1 Analysis of the interaction of garlic with mediators of inflammation involved in the molecular mechanism of atherosclerosis

  2. Year 1 and 2 • Influence of „garlic“ on the activation of the • transcription factor NF-kappaB • Influence of „garlic“ on the expression of adhesion • molecules (ICAM-1, VCAM-1, E-Selectin) • Influence of „garlic“ on cytokine production in blood cells

  3. Methods Cell models: HUVECs: human umbilical vein endothelial cells HEK 293: human embryonic kidney cell line

  4. Transcription factor NF-kB activation P proinflammatory factors i.e. CAM, Cytokines TNF-a P IkB NF-kB binding activity transactivation activity

  5. + radio-labeled oligonucleotides isolation of nuclear protein gel-electrophoresis autoradiography EMSA: electrophoretic mobility shift assay

  6. Parthenolid Diallyl disulfide 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 • 10 ng/ml TNF + cold NF-B • 10 ng/ml TNF + cold AP-2 • Untreated cells • 10 ng/ml TNF • 20 µM DADS without TNF • 5 µM DADS + TNF • 10 µM DADS + TNF • 20 µM DADS + TNF • 10 ng/ml TNF + cold NF-B • 10 ng/ml TNF + cold AP-2 • Untreated cells • 10ng/ml TNF • 20µM Par without TNF • 5µM Par + TNF • 10µM Par + TNF • 20µM Par + TNF EMSA results

  7. NF-kappaB luciferase reporter-gen assay luciferase TNF-a lysis activation luciferase light + substrate luciferase reporter- gen Luminometer

  8. Luciferase reporter-gen assay results without TNF + 1ng/ml TNF

  9. Luciferase reporter-gen assay results

  10. Summary No influence on NF-B translocation or activity had: • 1. Garlic constituents • S-allylcysteine • Allyl mercaptan • Diallyl sulfide • Diallyl disulfide • Allylmethyl-sulfoxide • -Glutamylcysteine • 2. Garlic dry powders • Printanor Year 2000 • Morasol Year 2000 • Morado Year 1999 • Printanor Year 2001 • Lichtwer garlic powder

  11. VCAM-1 ICAM-1 E-Selectin TNF-a NF-kappaB

  12. Measurement of adhesion molecules Analysis by FACS (fluorescence activated cell sorting) Fluorochrome-labeled antibodies against Test compound e.g. ICAM-1, VCAM-1, E-Selectin TNF-a ICAM-1 control TNF-a/ 8 h Endothelial cells (HUVECS)

  13. FACS Analysis of E-selectin 104,10 96,68 87,42 95,43 mean fluorescence 3,62 3,62

  14. FACS Analysis of E-selectin 72,03 73,86 73,69 69,15 mean fluorescence 5,67 6,24

  15. FACS Analysis of E-selectin

  16. Summary of FACS Analysis • No effect on the TNF induced expression of adhesion molecules were found for: • Allyl mercaptan • Diallyl disulfide • S- allylcysteine • Lichtwer garlic powder

  17. cytokine Cytokines / Chemokines (blood cells) supernatant: blood medium test compound = garlic LPS 37°C, 5% CO2,16 h ELISA (Enzyme –Linked-ImmunoSorbent-Assay)

  18. Il-1ß DADS (n=4) Il-10 DADS (n=4) Garlicconstituents • Allicin, AMSO2 and -Glutamylcysteine had no significant effect • DADS showed dose dependent reduction for Il-1 and Il-10

  19. DMSO extracts Printanor 2000 DMSO extracts Printanor 2001 -10 ng/ml LPS + 10 ng/ml LPS -10 ng/ml LPS + 10 ng/ml LPS TNF

  20. DMSO extracts Printanor 2000 DMSO extracts Printanor 2001 -10 ng/ml LPS + 10 ng/ml LPS -10 ng/ml LPS + 10 ng/ml LPS Il-1

  21. DMSO extracts Printanor 2000 DMSO extracts Printanor 2001 Il-10 [pg/ml Blood] Il-10 [pg/ml Blood] -10 ng/ml LPS + 10 ng/ml LPS -10 ng/ml LPS + 10 ng/ml LPS Il-10

  22. Printanor 0 Printanor 200 Effect of reduced cytokine levels

  23. Summary • NF-B and NF-B regulated genes are not the main target of garlic • Cytokine – release is modulated by garlic

  24. Outlook • Look at other proinflammatory transcription factor • Chemically characterized material is urgently needed

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