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探討基隆山葡萄之抽取物對氣球擴張引起 新生內膜增生之調控機轉

探討基隆山葡萄之抽取物對氣球擴張引起 新生內膜增生之調控機轉. 吴 介信 中国医药大学药学系（台湾） 中图分类号： R914 文献标识码： A 文章编号： 1818 － 0086 （ 2009 ） 01. INTRODUCTION :. Angioplasty has been routinely used in many patients with stable and unstable angina and acute myocardial infarction. Landau C et al.,1994

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探討基隆山葡萄之抽取物對氣球擴張引起 新生內膜增生之調控機轉

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  1. 探討基隆山葡萄之抽取物對氣球擴張引起新生內膜增生之調控機轉探討基隆山葡萄之抽取物對氣球擴張引起新生內膜增生之調控機轉 吴介信 中国医药大学药学系（台湾） 中图分类号：R914 文献标识码：A 文章编号：1818－0086（2009）01

  2. INTRODUCTION : • Angioplasty has been routinely used in many patients with stable and unstable angina and acute myocardial infarction. Landau C et al.,1994 • Restenosis remains an important and significant problem , and 20% to 50% of them develop significant recurrent stenosis within 6 months. Michel E.Bertrand et al.,1997 • 20%~30% of patients are still affected by restenosis after coronary stenting. Sturek and Reddy,2002

  3. MECHANISMS OF RESTENOSIS Remodeling : positive remodeling (adaptative or expansive remodeling) negative remodeling (constrictive or restrictive remodeling) adventitial fibrosis Mintz et al.,1996 Neointimal hyperplasia : stimulation, proliferation, and migration of smooth muscle cells. Michel E.Bertrand et al.,1997 Thrombosis : platelets and soluble coagulation factors promot smooth muscle cell migration and proliferation. Le Breton H et al .,1996; Ruggeri Z ,1994

  4. AIM: To explore the differential protein expression in response to angioplasty for developing an effective therapeutic remedy in preventing this devastating disease for restoring the living quality of these patients.

  5. METHOD : Angioplasty to R’t Common carotid a. 12 weeks 300- 400g male S. D rat 2h / 2D / 7D /14D/control ( 30隻x4=120, control=20 ) H&E stain 2D / Mass

  6. RESULT: 2h 2D

  7. 2D Control 2h 1 2 1 1 1 1 2 2 2 2 3 3 3 3 3 4 4 5 5 6 4 6 4 4 14D 7D 1: PDIA3 precursor 2: GLUD 1 3: PGK 1 4: GSTp2 1 1 2 2 3 3 4 4

  8. GLUD1 GSTp2 Hypoxia PDIA3 Calreticulin PGK1 insulin JNK MEK IGFR MMP Free radical FAK ERK Cell survival / Proliferation Apoptosis Migration / Remodeling Restenosis

  9. GLUD1 • Mt. matrix enzyme • Function: (1) nitrogen and Glutamate metabolism and the energy homeostasis (2) ammonia detoxification Mavrothalassitis rt al.,1988 (3) insulin secretion mechanisms Sener A et al.,1980 • Insulin or IGF-1 activated thePI3K/Akt and ERK pathway in the regulation of anti-apoptosis and proliferation of VSMC Jung F et al.,2000 • Insulin/insulin-like growth factor I (IGF-I) is unique among growth factors in promoting VSMC differentiation via preferential activation of phosphatidylinositol 3-kinase (PI3K) and Akt Bennett WL et al.,2007 • FAK phosphorylation and cell adhesion were reduced by PI3K inhibition Campbell M et al.,2005

  10. GLUD1 insulin MEK IGFR FAK ERK Cell survival / Proliferation

  11. Formation of ADP to ATP and vice versa When tumour cells become hypoxic , they make PGK to facilitate anaerobic production of ATP and angiogenesis activators such as VEGF R.H. Wenger,2002 HIF-1α can transcriptionally activate the PGK1 Stefan Kress et al.,1998 VEGF expression is regulated by cellular hypoxia mediated by HIF-1α under normal physiological conditions and possibly under pathological situations,such as tumorigenesis Forsythe JA et al.,1996;Randy L. Jensen et al.,2006 PGK 1

  12. Hypoxia HIF-1α PGK1 VEGF Cell survival / Proliferation

  13. GSTp2 • pi class of GST family (glutathione S-transferase) • GSTp detoxifies electrophiles by catalyzing their conjugation with reduced glutathione Hayes, J. D.,1995 • negative regulator of the MAPK pathway through protein–protein interactions with JNK Victor Adler et al.,1999

  14. GSTp2 JNK Free radical Cell survival / Proliferation Apoptosis

  15. PDIA3 • PDI family (protein disulfide isomerase) • Resident in the ER • Function : (1) form disulfide bonds and shuffle incorrect disulfides into their correct pairings Ellgaard, L.et al.,2003 (2) with calnexin and calreticulin to modulate glycoprotein folding John G. Elliott et al.,1997 (3) play a structural role in the MHC class I complex with TAP Garbi et al., 2006 • Calreticulin plays an important role in cardiac development and regulating MMP activity Wu M.2007

  16. PDIA3 Calreticulin MMP Migration / Remodeling

  17. GLUD1 GSTp2 Hypoxia PDIA3 Calreticulin PGK1 insulin JNK MEK IGFR MMP Free radical FAK ERK Cell survival / Proliferation Apoptosis Migration / Remodeling Restenosis

  18. Wild grape • 古稱蘡奧 • 原生長在中國大陸及台灣平原與山麓灌木叢林 • 唐朝《新修本草》,明朝《本草綱目》,清朝《植物名實圖考》, 《泉州本草》, 《江蘇藥材誌》 • 山葡萄在傳統中藥之中屬高經濟藥材，亦為台灣民間珍貴的保健作物，全株皆可利用，有補腎、明目、祛風、解毒、補血之功效 鍾錠全，1997 • 山葡萄粗抽物含有β-sitosterol, β-sitosteryl glycoside, (+)-catechin, ampelopside,gallic acid, β-amyrin,vanillic acid, quecertin, betulin, ethyl gallate, kaempferol, resveratrol • 功用: (1)瘡毒 (2)風疹 (3)抗血小板凝集 (4)鎮痛抗發炎 (5)清除自由基 (6)預防心血管疾病

  19. Inhibition of Lewis lung cancer growth by heyneanol A from the roots of Vitis amurensis through caspase-3 , caspase-9 and PRAP Lee EO et al.,2006 Inhibition of S1P- and VEGF-mediated migration of endothelial cell , HT-1080 , U-87, and DAOY by red grape skin polyphenolic extract Barthomeuf C et al.,2006 Grape seed extract had antiproliferative , apoptotic and inhibition of MMP2 secretion in HUVEC Agarwal C et al .,2004 Regulation of proliferation and gene expression in SMC by resveratrol and standardized grape extracts Zhirong Wang et al.,2006

  20. Vitis kelungeusis (VK) • 葡萄屬(vitis)多年生木質落葉性藤本植物 • The crude extract and its derived soluble fractions showed good antioxidant activities. Kai-Chung Cheng et al., National Chung-Hsing

  21. Stem ofVK Crude layer Water layer EtOAc layer (EA) Water layer (H2O) BuOH layer

  22. A10 ( Rat thoracic aorta smooth muscle cell ) Crude (15%FBS) EA (15%FBS) BuOH (15%FBS) H2O (15%FBS) Control (15% FBS) 24h MTT assay ( 590nm )

  23. MTT assay EA Crude H2O BuOH

  24. IC50 of the crude extract IC 50 : 0.13mg/ml

  25. IC50 of the EA extract IC 50 : 0.15mg/ml

  26. A10 ( 15%FBS -----> proliferation/migration/survival ) Crude 0.13mg/ml EA 0.15mg/ml BuOH 0.15mg/ml H2O 0.15mg/ml Control 24h WESTERN blot Chamber

  27. Migration-2 Control Angioplasty 2D 14D 2h 7D 0 MMP9 Pro form MMP9 active form MMP2 Pro form MMP2 active form

  28. Migration-2 Control Crude BuOH EA H2O Pro-MMP3 Act-MMP3

  29. DPPH Vit C Crude EA BuOH H2O 30 min DPPH assay ( 517nm )

  30. DPPH scavenging activity

  31. H2O2 Vit C Crude EA BuOH H2O 30 min Ex/Em = 530/590 nm

  32. H2O2 scavenging activity

  33. SUMMARY • GLUD1 . PGK1 . GSTp2 . PDIA3 play important roles in restenosis. • Crude and EA layers of VK extraction can affect SMC survival and proliferation. The most powerful one is EA layer. • All of VK extraction could affect active form of MMP3 activity and the most powerful one is EA layer. • According to the results , VK may be used as a potential drug to prevent restenosis.

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