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Kevin KM Yue 25th November 2006

Altered Expression of C-Reactive Protein in Ginsenoside-Re-Treated Diabetic Rats Detected by SELDI-TOF MS. Kevin KM Yue 25th November 2006. Diabetes Mellitus. Characterized by hyperglycemia and long-term complications of eyes, kidneys, nerves and blood vessels.

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Kevin KM Yue 25th November 2006

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  1. Altered Expression of C-Reactive Protein in Ginsenoside-Re-Treated Diabetic Rats Detected by SELDI-TOF MS Kevin KM Yue 25th November 2006

  2. Diabetes Mellitus • Characterized by hyperglycemia and long-term complications of eyes, kidneys, nerves and blood vessels. • ~180M diabetics in 2000, doubled by 2030

  3. Long term Hyperglycemia Leads to Serious Complications • Macrovascular • Coronary Heart • Disease • Cerebrovascular • accident • Peripheral Artery Diseases • Microvascular • Retinopathy • Nephropathy • Neuropathy

  4. Oxidative Stress plays major role in diabetic complications • Almost all diabetic complications associated with vascular dysfunctions of different tissues. • Oxidative stress implicated in pathogenesis of diabetes mellitus. • Common markers measured: GSH - primary endogenous antioxidant. MDA - marker of oxidative stress.

  5. Oxidative Stress plays major role in diabetic complications (L)Time course study of GSH concentration in Aorta of control & diabetic rats. (R) Time course study of MDA concentration in Aorta of control & diabetic rats. Results expressed as means ± SD (n=5). Statistical significance: P < 0.01 control compared with the diabetic group. Yue, K.K.M. et al. (2003). Life Sciences , 73, 2557-2570.

  6. Study effect of Ginsenoside-Re in diabetic rats

  7. Ginsenoside Re (人參皂甙Re) • Ginsenoside Re:One of the majoractive components of Panax ginseng; • Ginsenoside Re playssignificant role in antihyperglycemic action. Attele AS, et al. 2002. Diabetes, 5,1851-1858.

  8. Animal Model Sprague Dawley rats were used: • Streptozotocin-induced diabetic rats • Treated with 5, 10, 20 mg ginenoside Re Twice STZ 12-day Re treatment MDA, GSH Assay Week 4 6 7 8

  9. Effect of ginsenoside Re on tissue GSH levels • *P < 0.05, §P < 0.01 • Ginsenoside-treated diabetic group compared with vehicle-treated diabetic group

  10. Effect of ginsenoside Re on tissue MDA levels *P < 0.05 Ginsenoside-treated diabetic group compared with vehicle-treated diabetic group

  11. Summary 1 • Ginsenoside Re can normalize GSH and MDA levels First report of antioxidant effect of ginsenoside Re Cho, W.C.S., Chung, W.S., Lee, S.K., Leung, A.W.N., Cheng, C.H.K. and Yue, K.K.M. (2006). European Journal of Pharmacology , 550, 173-9.

  12. Time course study of potential biomarkers in diabetic rats by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF MS)

  13. Proteomic profiling • SELDI technology performs protein separation based on analytes’ surface property; • Proteinchip array acts as surface sample binds uniformly and matrix placed on chip only after proteins bound to chip; • By binding sample to chips with differing surface chemistries in parallel and in series, complex mixture of proteins reduced to sets of proteins with common properties.

  14. Proteinchip Technology: Proteinchip Array Method Step 1: Complex protein sample is placed on a ProteinChip Array • Affinity Capture – Proteins bind to chemical or biological sites on the ProteinChip surface Step 2: Remove unbound proteins • Wash the ProteinChip with appropriate stringency buffer • Bound proteins are retained (RETENTATE CHROMATOGRAPHY) Step 3: Add energy absorbing molecules or “Matrix”, MS detection • EAM is applied to facilitate desorption and ionization in the TOF-MS Chip Reader.

  15. Spectra View 7.5 5 4000 2000 2.5 0 Map View 6 8000 4730.4+H 6000 4618.2+H 4 2185.8+H 5045.2+H 2781.9+H 3915.2+H 2528.2+H 2 4000.2+H 3172.3+H 2369.9+H 7977.1+H 4345.6+H Laser Pixel Gel View 2000 4000 6000 8000 2000 4000 6000 8000 SELDI Proteinchip Technology-LDI-TOF-MS Detection • Retained proteins “eluted” from ProteinChip Array by Laser Desorption and Ionization • Ionized proteins detected and their mass accurately determined by Time-of-Flight Mass Spectrometry Laser Detector TOF-MS

  16. Animal Model Sprague Dawley rats were used: • Streptozotocin-induced diabetic rats: STZ 4 8 12 Week

  17. Potential biomarkers - Diabetes vs normal controls

  18. Proteomic profiling ofginsenoside Re-treated diabetic rats

  19. 8 potential biomarkers

  20. Changes of protein expression induced by treatment with ginsenoside Re Among the 8 potential biomarkers, normalized peak intensities at 25,442 Da significantly decreased after ginsenoside treatment in diabetic rats. Scatter plot of thebiomarker at 25,442 Da (A) (B) (C) (D) (E) (F)

  21. Protein matching • Potential biomarker at 25,442 Da matched C-reactive protein (CRP) in Swiss-Prot knowledgebase. • Further searching by molecular size and pI, fragment of CRP at MW 23,285 Da found to match with one of our significant differential peaks at 23,306 Da. • CRP: - indicator of inflammation; - High level of CRP generally associated with development of atherosclerosis and coronary heart disease.

  22. Concentration of C-reactive protein (ug/mL)

  23. SELDI + 2D PAGE + MALDI 4 serum biomarkers found by SELDI-TOF MS identified by 2D PAGE and MALDI-TOF MS as: • Ig Kappa Chain C region, B allele (KACB) • C-reactive protein (CRP) • T-kininogen I (KNT1) • Serotransferrin (TRFE)

  24. Identified proteins by2D PAGE and MALDI-TOF MS

  25. 12% gel, pH 4~7 Normal DM KNT1 HEMO HEMO KNT1 FETUA FETUA APOA4 APOA4 TRFE TRFE ITIH4 ITIH4 CRP CRP RETBP RETBP APOA1 APOA1 HPT TTHYD HPT TTHYD KACB KACB Proteins in RED – More abundant Proteins in GREEN – Less abundant

  26. Summary • CRP up-regulated in diabetes • Ginsenoside Re normalize CRP level in diabetic rats • Ginsenoside Re may improve diabetes and its complications by alleviation of inflammation Cho, W.C.S., Yip, T-T., Chung, W-S., Lee, S.K.W., Leung, A.W.N., Cheng, C.H.K. and Yue, K.K.M.. (2006). Journal of Ethnopharmacology , 108, 272-279.

  27. Acknowledgements • Dr. William CHO Chi Shing • Mr. CHUNG Wai Shing • Ms. LEE Kin Wah Collaborators: • Prof. CHENG Hon Ki Christopher • Dr. YIP Tai Tung

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