石斛菲醌衍生物抗肝纖維化活性之研究

Study of Dendrobium phenanthrenequinone derivatives in anti-hepatic fibrosis

10.6834/CSMU.2014.00093

陳姿惠

石斛屬 ； 肝纖維化 ； 轉型生長因子-β1 ； 腫瘤壞死因子-α ； Denbinobin ； Moniliformediquinone ； Dendrobium SWARTZ ； Hepatic fibrosis ； Transforming growth factor-β1 ； Tumor necrosis factor-α

中山醫學大學應用化學系碩士班學位論文

2014年

碩士

曾翠華

繁體中文

石斛屬(Dendrobium SWARTZ)，隸屬於蘭科（Orchidaceae）植物，生長於高山溼潤之巖石或古木上。傳統中藥材石斛，多採用金釵石斛（Dendrobium Nobile Lindl.）、銅皮石斛 (Denbinobin moniliform)，或其多種同屬植物的莖為基原，早期文獻中也指出石斛具有抗氧化、抗發炎以及抑制腫瘤生成等藥理活性，然而有關於石斛菲醌類成分之活性研究卻仍是鮮少有較明確深入的探討結果。本研究透過合成方式取得銅皮石斛 (Denbinobin moniliform)中之菲醌成份Denbinobin及其衍生物 Moniliformediquinone (以下簡稱 MFD ) ，進行研究探討是否可以抑制肝纖維化之作用。 本研究初步使用肝臟星狀細胞株(HSC-T6)和正常小鼠肝細胞（BNL CL.2）進行體外實驗以 Denbinobin 和 MFD 處理細胞，透過細胞毒性分析(MTT assay)及細胞增生分析(BrdU assay)，偵測 Denbinobin 和 MFD 對細胞增生及毒性的影響，接著以DAPI染色、F-actin染色和AnnexinV-FITC/PI 雙染法分析MFD處理過後肝星狀細胞凋亡的現象和形態上的變化，並以西方墨點法分析細胞凋亡相關蛋白 Bcl-2 family，發炎相關蛋白包含轉化生長因子-β1（TGF-β1）、腫瘤壞死因子-α(TNF-α)和細胞間粘附分子-1（ICAM-1），及促纖維化相關蛋白包含平滑肌α-肌動蛋白（α-SMA），COL-1、CTGF、GFAP 和 TIMP-1 等蛋白表現以探討其分子機制。研究結果顯示，發現兩者皆能有效抑制肝星狀細胞(HSC-T6 cell)增生，但 MFD 卻對正常小鼠肝細胞（BNL CL.2）呈現較低毒性，細胞凋亡的現象隨著濃度上升凋亡的現象也就越顯著，也發現MFD會促進Bax表現，且負調控Bcl-2 family，在F-actin染色結果中亦可發現在形態上隨著MFD濃度上升，可降低偽足與觸角的生成或改變其形態，達到抑制活化的星狀細胞之遷移與沉積產生之纖維化現象。此外，利用西方墨點法探討纖維化之現象及分子機制，經MFD處理過的星狀細胞顯著的降低COL-1、CTGF、GFAP 和 TIMP-1 等促纖維化相關蛋白。在體內實驗中，利用四氯化碳(CCl4)誘導小鼠肝纖維化作用，期間以 0.1 mg/kg 及 0.5 mg/kg MFD 做治療。實驗結果顯示， MFD 顯著降低血液生化指標中天冬氨酸轉氨酶（AST），丙氨酸轉氨酶（ALT）和乳糖脫氫酶（LDH）以及肝組織中促發炎、纖維化相關蛋白如 TNF-α、TGF-β1，α-SMA 和 Col-1 等表現。透過病理分析，也發現到 MFD 可減輕四氯化碳誘導的肝毒性和肝纖維化。這些結果表示了 MFD 具有治療肝纖維的化潛力。

Plants of Dendrobium genus (Orchidaceae) are used in traditional herbal medicines in Asia, and studies have shown that they cotain a wide variety of medicinal properties including antioxidant, anti-inflammation and anti-cancer. In the present study, the anti-hepatic fibrosis potential of synthesized moniliformediquinone (MFD) and denbinobin, which have been isolated from Dendrobium moniliforme, are investigated. Since activated hepatic stellate cells are considered to be an essential part of hepatic fibrosis, the effects of MFD and denbinobin on activated hepatic stellate cells were evaluated first. The cytotoxicity of MFD and denbinobin in hepatic stellate cell line (HSC-T6) and normal mouse liver cells (BNL CL.2) was determined by MTT assay. It showed that both MFD and denbinobin inhibited cell viability of HSC-T6 cells in a dose dependent manner, but the MFD showed low toxicity in BNL CL.2cells. MFD also induced apoptosis and inhibited proliferation in HSC-T6 cells by Annexin V-FITC/PI double staining analysis and BrdU incorporation assay respectively. By western immunoblotting analysis, MFD and denbinobin up-regulated the expression of Bax and down-regulated the expression of Bcl-2. Furthermore, MFD and denbinobin inhibited the expression of inflammation-related proteins such as transforming growth factor -β1 (TGF-β1), tumor necrosis factor -α (TNF-α) and intercellular adhesion molecule - 1 (ICAM-1), and the expression of pro-fibrotic related proteins containing smooth muscle α- actin (α-SMA), COL-1, CTGF, GFAP and TIMP-1. In vivo, liver fibrosis was induced by administration of CCl4 twice weekly for 10 weeks in mice. The administration of MFD thrice weekly was started after 1 week of CCl4 administration. MFD significantly reduced the plasma aspartate transaminase (AST), alanine trasaminase (ALT) and lactose dehydrogenase (LDH) as well as hepatic expression of TGF-β1, α-SMA, and Col-1 in CCl4 –injected mice. By pathological analysis stained with H&E and Masson, MFD alleviated CCl4-induced hepatic toxicity and fibrosis. These results implicate that MFD possesses therapeutical potential for liver fibrosis.

1. Arthur M.J. Fibrogenesis II Metalloproteinases and their inhibitors in liver fibrosis. American Journal of Physiology - Gastrointestinal and Liver Physiology, (279):245-249, 2000.
   連結：
2. Baud, V., and Karin, M. Signal transduction by tumor necrosis factor and its relatives. Trends in Cell Biology Journal, (9):372-377, 2001.
   連結：
3. Bissell DM., Roulot D., George J. Transforming Growth Factor βand the liver. Hepatology, (34):859-867, 2001.
   連結：
4. Burt AD. Pathobiology of hepatic stellate cells. Journal of Gastroenterology, (34):299-304, 1999.
   連結：
5. Caligiuri A., Defranco R., Romanelli RG., Gentilini A., Meucci M., Failli P., Mazzetti L. Antifibrogenic effect of canrenone, an antialdosteronic drug, on human hepatic stellate cells. Gastroenterology, (124):504-520, 2003.
   連結：
6. Chen A., Zhang L., Xu J., Tang J. The antioxidant (-)-epigallocatechin-3-gallate inhibits activated hepatic stellate cell growth and suppresses acetaldehyde-induced gene expression. Biochemical Journal, (368):695-704, 2002.
   連結：
7. Chen P.H., Peng C.Y., Pai H.C., Teng C.M., Chen C.C., Yang C.R. Denbinobin suppresses breast cancer metastasis through the inhibition of Src-mediated signaling pathways. Journal of Nutritional Biochemistry, (22):732-740, 2011.
   連結：
8. Chien-Chih C., Li-Gin W., Feng-Nien K., Che-Ming T. Antiplatelet aggregation principles of Dendrobium loddigesii. Journal of Natural Products, (9):271-274, 1994.
   連結：
9. Chilakapati J., Shankar K., Korrapati M.C., Hill R.A., Mahendale H.M. Saturation toxicokinetics of thioacetamide: role in initiation of liver injury. Drug Metabolism and Disposition, (33):1877-1885, 2005.
   連結：
10. Cohen G.M. Caspases: the executioners of apoptosis. Biochemical Journal, (326):1-16, 1997.
    連結：
11. Czaja M.J., Weiner F.R., Flanders K.C., Giambrone M.A., Wind R.,Chen Y.M., Wu K.D., Tsai T.J., Hsieh BS. Pentoxifylline inhibits PDGF-induced proliferation of and TGF-beta-stimulated collagen synthesis by vascular smooth muscle cells. Journal of Molecular and Cellular Cardiology, (4):773-83, 1999.
    連結：
12. Ebada S.S., Edrada R.A., Lin W., Proksch P. Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates. Nature Protocols, (3):1820-1831, 2008.
    連結：
13. Failli P., Defranco R., Caliguiri A., Gentilini A., Romanelli RG. Nitrovasodilators inhibit Platelet-derived growth factor-induced proliferation and migration of activated human hepatic stellate cells. Gastroenterology, (119): 479-492, 2000.
    連結：
14. Friedman S.L. Liver fibrosis – from bench to bedside. Journal of Hepatology, (38):38-53, 2003.
    連結：
15. Friedman S.L. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. Journal of Biological Chemistry, (275): 2247-2250, 2000.
    連結：
16. Ghobrial I.M., Witzig T.E., Adjei A.A. Targeting Apoptosis Pathways in Cancer Therapy. Cancer Journal for Clinicians, (55):178-194, 2005.
    連結：
17. Gressner A.M., Weiskirchen R., Breitkopf K., Dooley S. Roles of TGF-beta in hepatic fibrosis. Frontiers in Bioscience, (7):793-807, 2002.
    連結：
18. Hinz B., Formation and function of the myofibroblast during tissue repair. Journal Invest Dermatol, (3):526-537, 2007.
    連結：
19. Hsu J.L., Lee Y.J., Leu W.J., Dong Y.S., Pan S.L., Uang B.J. and Guh J.H. Moniliformediquinone Induces In Vitro and In Vivo Antitumor Activity through Glutathione Involved DNA Damage Response and Mitochondrial Stress in Human Hormone Refractory Prostate Cancer. The Journal of Urology, (191):1429-1438, 2014.
    連結：
20. Iimuro Y., Gallucci R. M., Luster M. I., Kono H., Thurman R. G. Antibodies to tumor necrosis factor alfa attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat. Hepatology, (6):1530-1537, 1997.
    連結：
21. Ishak K, Baptista A, Bianchi L, et al. Histologic grading and staging of chronic hepatitis. Journal of Hepatology, (24):289-293, 1995.
    連結：
22. Inoue T, Jackson EK. Strong antiproliferative effects of baicalein in cultured rat hepatic stellate cells. European Journal of Pharmacology (378):129-135, 1999.
    連結：
23. Jeong H. K., Su Y. O., Sang B. H., Golam M. U., Chul Y. K., Jae K. L. Anti-inflammatory effects of Dendrobium nobile derived phenanthrenes in LPS-stimulated murine macrophages. Archives of Pharmacal Research, (10):253-269, 2014.
    連結：
24. Jui-Ling H., Yean-Jang L., Wohn-Jenn L., Yu-Shun D., Shiow-Lin P., Biing-Jiun U., Jih-Hwa G. Moniliformediquinone Induces In Vitro and In Vivo Antitumor Activity through Glutathione Involved DNA Damage Response and Mitochondrial Stress in Human Hormone Refractory Prostate Cancer. The Journal of Urology, (191):1429-1438, 2014.
    連結：
25. Kim KY., Rhim TY., Choi I., Kim SS. N-Acetylcysteine induces cell cycle arrest in hepatic stellate cell through its reducing activity. Journal of Biological Chemistry (276): 40591-40598, 2001.
    連結：
26. Knodell R.G., Ishak K.G., Black W.C., Chen T.S., Craig R., Kaplowitz N., Kiernan T.W., Wollman J., Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Journal of Hepatology, (5):431-435, 1981.
    連結：
27. Latha S., Phillip HG., Mark EB., Sandra J. and Sandeep K. Reversal of TGF-b1 stimulation of a-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren’s-derived fibroblasts. BioMed Central Musculoskeletal Disorders, (12):113, 2011.
    連結：
28. Lechuga C.G., Hernández-Nazara Z.H., Domínguez Rosales J.A., Morris E.R., Rincón A.R., Rivas-Estilla A.M., Esteban-Gamboa A., Rojkind M. TGF-β1 modulates matrix metalloproteinase-13 expression in hepatic stellate cells by complex mechanisms involving p38MAPK, PI3-kinase, AKT, and p70S6k. American Journal of Physiology - Gastrointestinal and Liver Physiology, (287): 974-987, 2004.
    連結：
29. Lin T. H., Chang S. J., Chen C. C., Wang J. P., Tsao L. T. TwoPhenanthraquinones from Dendrobium moniliforme. Journal of Natural Products, (8):1084-1086, 2001.
    連結：
30. Li T., Wang J., Lu Z. Accurate identification of closely related Dendrobium species with multiple species-specific gDNA probes. Journal of Biochemical and Biophysical Methods, (2):111-23, 2005.
    連結：
31. Lin YL., Wu CH., Luo MH., Huang YJ., Wang CN., Shiao MS., Huang YT. In vitro protective effects of salvianolic acid B on primary hepatocytes and hepatic stellate cells. Journal of Ethnopharmacology, (105):215-222, 2006.
    連結：
32. Maher J. J., McGuire R. F. Extracellular matrix gene expression increase preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo. Journa Clin Invest, (86): 1641-1648, 1990.
    連結：
33. Nelson D. L. and Cox M. M. Lehninger's Principles of Biochemistry. 4th Edition, W. H. Freeman and Company, 2005.
    連結：
34. Nakatani T., Honda E., Hayakawa S., Sato M., Satoh K., Kudo M., Munakata H. Effects of decorin on the expression of alpha-smooth muscle actin in a human myofibroblast cell line. Molecular and Cellular Biochemistry, (1-2):201-207, 2008.
    連結：
35. Olaso E, Friedman SL. Molecular regulation of hepatic fibrogenesis. Journal of Hepatology, (29):836-847, 1998.
    連結：
36. Palanki M. S. Inhibitors of AP-1 and NF-kappa B mediated transcriptional activation: therapeutic potential in autoimmune diseases and structural diversity. Current Medicinal Chemistry, (2):219-227, 2002.
    連結：
37. Schmierer B., Hill CS. Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor b-dependent nuclear accumulation of Smads. Molecular and Cellular Biology, (25):9845-9858, 2005.
    連結：
38. Senthil K.R., Claudine S.F., Feridoon N., Andrea H.,Eugene S.K., Angela L.T., and Andrei L.G. Constitutive expression of E2F-1 leads to p21-dependent cell cycle arrest in S phase of the cell cycle. Oncogene, (23):4173-4176, 2004.
    連結：
39. Senoo H., Imai K., Matano Y., Sato M. Molecular mechanisms in the reversible regulation of morphology, proliferation and collagen metabolism in hepatic stellate cells by the three-dimensional structure of the extracellular matrix. Journal of Gastroenterology and Hepatology, (13):19-32, 1998.
    連結：
40. Shiha G. and Zalata K. Ishak versus METAVIR: Terminology, Convertibility and Correlation with Laboratory Changes in Chronic Hepatitis C. Liver Biopsy, (10):156-168, 2011.
    連結：
41. Song J. I., Kang Y. J., Yong H.Y., Kim Y. C. and Moon A. Denbinobin, a phenanthrene from Dendrobium nobile,inhibits invasion and induces apoptosis in SNU-484 human gastric cancer cells. Oncology Reports, (3):813-818, 2012
    連結：
42. Suematsu M and Asio S Professor Toshio Ito: a clairvoyant in pericyte biology. Keio Journal of Medicine, (50):66-71, 2001.
    連結：
43. Szpak, Paul. Fish bone chemistry and ultrastructure: implications for taphonomy and stable isotope analysis. Journal of Archaeological Science, (12):3358–3372, 2011.
    連結：
44. Tilg H, Kaser A, Moschen AR. How to modulate inflammatory cytokines in liver diseases. Liver International, (26):1029-1039, 2006.
    連結：
45. Totsukawa G,Wu Y, Sasaki Y, Hartsborne DJ, Yamakita Y, Yamashiro S,Matsumura F. Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts. Journal of Cell Biology, (164):427-439, 2004.
    連結：
46. Tsukada S., Parsons C.J., Rippe R.A. Mechanisms of liver fibrosis. Clinica Chimica Acta, (364):33-60, 2006.
    連結：
47. Wrigley TC. Ayapin, scopoletin and 6,7-dimethoxycoumarin from Dendrobium thyrsiflorum (Reichb. fil.). Nature, (188):1108, 1960.
    連結：
48. Yang HK., Lee PJ., Jeong EJ., Kim HP. and Kim YC., Selective Apoptosis in Hepatic Stellate Cells Mediates the Antifibrotic Effect of Phenanthrenes from Dendrobium nobile. Phytotherapy Research, (26): 974-980, 2012.
    連結：
49. Zamara E, Novo E, Marra F, Gentilini A, Romanelli RG, Caligiuri A, Robino G, et al.4-Hydroxynonenal as a selective pro-fibrogenic stimulus for activated human hepatic stellate cells. Journal of Hepatology, (40):60-67, 2004.
    連結：
50. Zhou SF, Kestell P. Thalidomide in cancer treatment: A potential for elderly. Drugs and Aging, (2): 85-100, 2002.
    連結：
51. Ahern M., Hall P., Halliday M., Liddle C., Olynyk C. Hepatic stellate cell nomenclature. Hepatology, (1):193, 1996.
52. Bradham, C.A., Plumpe, J., Manns, M.P., Brenner, D.A., and Trautwein, C. Mechanisms of hepatic toxicity. I. TNF-induced liver injury. physiology journal, (275):387-392, 1998.
53. Gorres K.L., Raines Ronald T. Prolyl 4-hydroxylase. Critical Reviews in Biochemistry and Molecular Biology, (2):106-124, 2010.
54. Junqueira L. C., Carneiro J., Basic Histology, Eleventh Edition, 2005.
55. Knittel T, Saile B, Ramadori G. Fibrogenesis. Pathophysiology and therapeutic approaches. Internist (Berl) (39):238-246, 1998.
56. Milani S., Herbst H., Schuppan D., Grappone C., Pallegrini G., Pinzani M., Casini A., Calabro A., Ciancio G. Differential expression of matrix-metalloproteinase-1and -2 genes in normal and fibrotic human liver. American Journal of Pathology, (144): 528-537, 1994.
57. Portmann B., Slapak GI., Gane E., Williams R. Pathology and biopsydiagnosis of the transplanted liver. Verhandlungen der Deutschen Gesellschaft fur Pathologie, (79):277-290, 1995.
58. Shen C., Jenny Y., Hongbao M. Alpha-Smooth Muscle Actin (α-SMA). The Journal of American Science, (4):7-9, 2008.
59. Su AI, Cooke MP., Ching KA., Hakak Y., Walker JR., Wiltshire T., Orth AP., Vega RG, Sapinoso LM., Moqrich A., Patapoutian A., Hampton GM., Schultz PG., Hogenesch JB. Large-scale analysis of the human and mouse transcriptomes. Proceedings of the National Academy of Sciences, (7):4465-4470, 2002.
60. Tzong-Huei L., Shu-Jen C., Chung-Chuan C., Jih-Pyang W., Lo-Ti T. Two Phenanthraquinones from Dendrobium moniliforme. Journal of Natural Products, (64):1084-1086, 2001.
61. Yong H., Chou G.X., Wang Z.T., Guo Y.W., Hu Z.-B., Xu L.S.. Two new compounds from Dendrobium chrysotoxum. Helvetica Chimica Acta,(87):394-399, 2004.
62. 馬國祥、徐國鈞、徐珞珊、王崢濤、菊池沏。鼓槌石斛中一新的聯卞化合物－兒鼓槌石斛素。藥學學報 (31): 222-225, 1996。
63. 黃民權、蔡體育、劉慶倫，鐵皮石斛多醣對小白鼠白細胞數翰林巴細胞移動抑制因子的影響。天然產物研究與開發 (3):39, 1996。