臺灣博碩士論文加值系統

本篇論文主要目的在於探討naphthaoquinone類衍生物，Methylnaphthazarin及Naphthazarin對血管張力的影響，及其參與的調控機制。我們利用離體的大鼠胸主動脈環張力實驗，發現在低濃度下，Methylnaphthazarin (0.1-10 mM) 及Naphthazarin (0.01-0.1 mM) 具有濃度關係的增加Phenylephrine (PE)所導致的血管收縮作用，且此作用必須在內皮細胞存在下才能發生，同時也發現Methylnaphthazarin及Naphthazarin具有濃度關係的抑制了Acetylcholine造成的血管放鬆作用，顯示兩者可能透過抑制NO-cGMP pathway的路徑而影響了血管的正常功能，並由以上結果推測內皮細胞在此扮演重要角色。
因此我們利用分離自人類臍靜脈的血管內皮細胞 (HUVECs)來探討抑制ACh造成血管放鬆的機制，發現Methylnaphthazarin及Naphthazarin具有濃度關係的抑制一氧化氮 (Nitric Oxide)的生成及一氧化氮合成酶 (Nitric Oxide Synthase, NOS)的活性，但並不影響eNOS蛋白質的表現。證實Naphthazarin及Methylnaphthazarin可能透過抑制一氧化氮的生成而導致對ACh引發的血管舒張反應的抑制。此外，我們亦初步發現Naphthazarin及Methylnaphthazarin可能會誘發Superoxide anion (O2-.)的生成。
綜合以上的結果，我們認為Methylnaphthazarin及Naphthazarin這兩種Naphthoquinone類衍生物可能透過抑制血管內皮細胞一氧化氮之活性，導致對於血管張力的增強及血管鬆弛的抑制。至於確實的作用機制與O2-.在此所扮演的角色可能需要更進一步的探討。

The purpose of this experiment is to investigate the effect of naphthoquinone derivatives, methylnaphthazarin and naphthazarin in rat thoracic aortic rings isolated from rats and the pathway involved. After incubated with rat thoracic aortic rings, we found methylnaphthazarin and naphthazarin concentration-dependently potentiated phenylephrine induced vasocontraction and inhibited acetylcholine induced vasorelaxation in endothelium dependent manner. These data suggested that endothelium might be involved in this pathway.
Human Umbilical Endothelium Vein Cells (HUVECs) were used to investigate the effect of naphthazarin and methylnaphthazarin on endothelium. We found methylnaphthazarin and naphthazarin concentration-dependently inhibited nitric oxide production and eNOS activity, with no effect on eNOS protein expression. These data suggested that the inhibitory effect of methylnaphthazarin and naphthazarin on vasorelaxation induced by acetylcholine might due to the inhibition of NO production and eNOS activity. We further detected the increasing of superoxide anion formation induced by naphthazarin and methylnaphthazarin.
Finally, we concluded methylnaphthazarin and naphthazarin might inhibit endothelium nitric oxide activity resulted in the potentiated vasocontraction and inhibited vasodilation.

目錄
中文摘要………………………………………………………………………………I
英文摘要……………………………………………………………………………...II
縮寫表……………………………………………………………………………….III
第一章 緒論
前人研究………………………………………………………………………………1
血管的生理功能……………………………………………………………………....4
1.血管張力調控機轉………………………………………………………...4
2.內皮細胞生理功能………………………………………………………...4
3.一氧化氮生理功能與相關疾病…………………………………………...5
4.一氧化氮合成酶…………………………………………………………...7
5.一氧化氮合成酶結構與活化機轉………………………………………...7
研究動機…………………………………………………………………………...….9
圖1. Quinone衍生物氧化還原反應途徑………………………………………...…10
結構圖………………………………………………………………………………..11
第二章 實驗材料與方法
實驗材料…………………………………………………….……………………….12
實驗方法
大鼠胸主動脈環製備與血管張力試驗……………………………………………..13
人類臍靜脈內皮細胞的分離與培養……………………………………………..…14
細胞毒性測試……………………………………………………………………......14
西方點墨法…………………………………………………………………………..15
一氧化氮(NO)含量測定…………………………………….………………...…….16
一氧化氮合成酶(NOS)活性測定………………………….…………………...…...17
Superoxide含量測定…..…………………………………………………….……...17
統計分析……………………………………………………..……………………....18
第三章 實驗結果
Naphthazarin和Methylnaphthazarin對phenylephrine引發的血管收縮
的影響.…………………………………………………………………………….19
Naphthazarin和Methylnaphthazarin對Acetylcholine引發的血管張力
之影響……………………………………………………………………………..19
Methylene blue及L-NAME對高濃度Methylnaphthazarin及Naphthazarin
所誘發血管收縮的影響…………………………………………………………..20
HUVECs對Methylnaphthazarin及Naphthazarin引發之細胞毒性測試
結果…………………………………………………………………………….….21
Naphthazarin及Methylnaphthazarin對HUVECs生成NO的影響……………21
Naphthazarin及Methylnaphthazarin對HUVECs上NOS活性的
影響……………………………………………………………………………..…22
Naphthazarin及Methylnaphthazarin對HUVECs上eNOS蛋白質
表現的影響…………………………………………………….………...….….......22
Naphthazarin及Methylnaphthazarin對HUVECs上superoxide 生成
影響…………………………………………………………………………………23
Fig.1 Typical tracings of methylnaphthazarin and naphthazarin on
the relaxation of rat thoracic aorta induced by acetylcholine…………..…….24
Fig.2 Effect of methylnaphthazarin and naphthazarin on rat thoracic aorta
after preincubated with L-NAME and methylene blue…………………….…...25
Fig.3 Dose response curve of phenylephrine in the present of
naphthazarin in rat thoracic aorta……………………………………..…….....26
Fig.4 Dose response curve of phenylephrine in the present of
methylnaphthazarin in rat thoracic aorta…………………….………..……...…27
Fig.5 Dose response curve of acetylcholine in the present of naphthazarin
in rat thoracic aorta………………………………………………………….…..28
Fig.6 Dose response curve of acetylcholine in the present of
methylnaphthazarin in rat thoracic aorta…………….……………....………..29
Fig.7 Result of cytotoxicity test……………………………………………….30
Fig.8 Effect of naphthazarin in the production of nitric oxide
in 20 min induced by A23187……………………………………………………31
Fig.9 Effect of methylnaphthazarin in the production of nitric oxide
in 20 min induced by A23187………………………………………...………….32
Fig.10 Effect of naphthazarin in the production of nitric oxide
in 24 hr induced by A23187…………………………………………………...…33
Fig.11 Effect of methylnaphthazarin in the production of nitric oxide
in 24hr induced by A23187…………………………………….……………….34
Fig.12 Inhibition of eNOS activity by naphthazarin……………………………...35
Fig.13 Inhibition of eNOS activity by methylnaphthazarin……………...………36
Fig.14 Expression of eNOS protein after incubated with naphthazarin
24 and 48 hr…………………………………………...……..………………...…37
Fig.15 Expression of eNOS protein after incubated with methylnaphthazarin
12 and 24 hr………………………………………………………..…………..…38
Fig.16 Effect of naphthazarin in the production of superoxide……………...…..39
Fig.17 Effect of methylnaphthazarin in the production of superoxide………….40
第四章 討論………………………………………………………………………..41
第五章 結論………………………………………………………………………....45
圖2. Methylnaphthazarin及Napththazarin可能作用途徑………………………….46
參考文獻……………………………………………………………………………..47

Angeliki, P.K., Andreana, N.A., Vassilios, P.P., Antonis, G., Panos, N.K. Alkannin and Shikonin: Effect on free radical processes and on inflammation-A preliminary pharmacochemical investigation. Arch. Pharm. Pharm. Med. Chem. 6:262-266, 2002.
Abusoud, H.M., Stuehr, D.J. Nitric oxide synthases reveal a role for calmodulin in controlling electron transfer. Proc. Natl. Acad. Sci. 90:10769-10772, 1993
Bradford, M.M. A rapid and sensitive method for the quantiation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.72: 248-254, 1976
Brunmark, A., Cadenas, E., Lind, C., Segura, A.J., Ernster, L. DT-diaphorase-catalyzed two-electron reduction of quinone epoxides. Free. Radic. Biol. Med. 3(3): 181-188, 1987
Carmichael, J., Degraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, J.B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47: 936-942, 1987
Choi, D.H., Kim, D.H., Park, Y.G., Chun, B.G., Choi, S.H. Protective effects of rilmenidine and agn 192403 on oxidative cytotoxicity and mitochondrial inhibitor-induced cytotoxicity in astrocytes. Free Radic. Biol. Med. 33:1321-1333, 2002
Choi, S.H., Choi, D.H., Lee, J.J., Park, M.S., Chun, B.G. Imidazoline drugs stabilize lysosomes and inhibit oxidative cytotoxicity in astrocytes. Free Radic. Biol. Med. 32: 394-405, 2002
Ciriolo, M.R., Martino, A.D., Lafavia, E., Rossi, L., Carri, M.T., Rotilio, G. Cu,Zn-Superoxide dismutase-dependent apoptosis induced by nitric oxide in neuronal cells. J. Biol. Chem. 275: 5065-5072, 2000
Cosentino, F., Still, J.C., Katusic, Z.S. Role of superoxide anions in the mediation of endothelium-dependent contractions. Hypertension 23: 229-235, 1994
De Vriese A.S., Verbenren, T.J., Van de, V.J., Lameire, N.H., Vanhoutte, P.M. Endothelial dysfunction in diabetes. Br. J. Pharmacol. 130(5): 963-974, 2000
Dodd, N.J.F. Mukherfee, T. Free radical formation from anthracycline antitumour. Biochem. Pharmacol. 33:379-385, 1984
Espino, P.C. and Sullivan, P.D. In vitro inhibition of the metabolism and mutagenicity of benzo (a) pyrene and benzo (a) pyrene-7,8-dihydrodiol by naphthazarin and other naphthol derivatives. Biochem. Biophys. Res. Commun. 142: 939-946, 1987
Feffeira, M.A., Costa, M.A.C., Alves, A.C. Identification of methylnaphthazarin in Diospyros Species. Phytochem. 11: 2352-2353, 1972
Forstermann, U., Mulsch, A., Bohme, E., Busse, R. Stimulation of soluble guanylate cyclase by an acetylcholine-induced endothelium-derived factor from rabbit and canine arteries. Circ. Res. 58:531-538, 1986
Forstermann, U., Closs, E.I., Pollock, J.S., Nakane, M., Schwarz, P., Gath, I., Kleinert, H. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23: 1121-1131, 1994
Galizzi, J.P., Qar, J., Fosset, M. Regulation of calcium channels in aortic muscle cells by protein kinase C activation ( diacylglycerol and phorbol esters) and by peptides (vawopressin and bombessin) that stimulate phosphoinositides breakdown. J. Biol. Chem. 262:6947-6950, 1987
Gruhn, N., Aldershvile, J., Boesgaard, S. Tetrahydrobiopterin improves endothelium-dependent vasodilation in nitroglycerin-tolerant rats. Eur. J. Pharmacol. 416: 245-249, 2001
Harrison, D., Griendling, K.K., Landmesser, U., Hornig, B., Drexler, H. Role of oxidative stress in atherosclerosis. Am. J. Cardiol. 91:7A-11A, 2003
Heller, R., Unbehaun, A., Schellenberg, B., Mayer, B., Werner-Felmayer, G., Werner, E.R. L-Ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin. J. Biol. Chem. 276: 40-47, 2001
Hemmens, B., Mayer, B. Emzymology of nitic oxide synthases. Methods Mol. Cell. Biol. 100:1-32, 1998
Hsu, K.S., Shiau-Lin, S.Y. Antagonism by paraquat of the relaxing effects of acetylcholine and A23187 in rat thoracic aorta. Eur. J. Pharmacol. 292:315-320, 1995
Huang, P.L., Huang, Z.H., Mashimo, H., Bloch, K.D., Moskowitz, M.A., Bevan, J.A., Fishman, M.C. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 377: 239-242, 1995
Jaffe, E.A., Nachman, R.L., Becker, C.G., Minick, C.R. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J. Clin. Invest. 52: 2745-2756, 1973
Jaffrey, S.R., Snyder, S.H. Nitric oxide : A neural messenger. Annu. Rev. Cell Dev. Biol. 11: 417-440, 1995
Kapadia, G.J., Balasubramanian, V., Tokuda, H., Konoshima, T., Takasaki, M., Koyama, J., Tagahaya, K., Nishino, H. Anti-tumor promoting effects of naphthoquinone derivatives on short term Epstein-Barr early antigen activation assay and in mouse skin carcinogenesis. Cancer Lett. 113: 47-53, 1997
Katusic, Z.S. Vascular endothelial dysfunction：does tetrahydrobiopyerin play a role？ Am. J. Physiol. Heart Circ. Physiol. 281: H981-H986, 2001
Li, H., Forstermann, U. Nitric oxide in the pathogenesis of vascular disease. J. Pathol. 190: 244-254, 2000
Likhitwitayawuid, K., Kaewamatawong, R., Ruangrungsi, N., Krungkrai, J. Antimalarial naphthoquinones from Nepenthes thorelii. Planta Med. 64: 237-241, 1998
Lim, H., Dey, S.K. Minireview：A novel pathway of prostacyclin signaling-Hanging out with nuclear receptor. Endocrinol. 143: 3207-3210, 2002
Menna, P., Salvatorelli, E., Giampietro, R., Liberi, G., Teodori, G., Calafiore, A.M., Minotti, G. Doxorubicin-dependent reduction of ferrlmyoglobin and inhibition of lipid peroxidation: Implications for cardiotoxicity of anticancer anthracyclines. Chem. Res. Toxicol. 15: 1179-1189, 2002
Min, B.S., Miyashiro, H., Hattori, M. Inhibitory effects of quinines on Rnase H activity associated with HIV-1 reverse transcriptase. Phytother. Res. 16: S57-S62, 2002
Minor, R.J., Myers, P.R., Guerra, R.J., Bates, J.N., Harrison, D.G., Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J. Clin. Invest. 86: 2109-2116, 1990
Ohta, A., Sivalingam, P.M., Lin, S., Ikekawa, N. Isolation of naphthazarin from walnut ‘Onigurumi’ and its inhibitory action on oxidative phosphorylation in mitochondria. Toxicon 11: 235-241, 1973
Ollinger, K. and Brunk, U.T. Cellular injury induced by oxidative stress is mediated through lysosomal damage. Free Radic. Biol. Med. 19: 565-574, 1995
Ollinger, K. and Brunmark, A. Effect of hydroxy substituent position on 1,4-Naphthoquionone toxicity to rat hepatocytes. J. Biol. Chem. 266: 21496-21503, 1991
Ollinger, K. Inhibition of cathepsin D prevents free-radical-induced apoptosis in rat cardiomyocytes. Arch Biochem Biophys. 373: 346-351, 2000
Petros, A., Bennett, D., Vallance, P. Effect of nitric oxide synthase inhibitors on hypotension in patients with septic shock. Lancet 338: 1557-1558, 1991
Rapoport, R.M., Murad, F. Agonist-induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through cGMP. Circ. Res. 52: 352-357, 1983
Roberg, K., Johansson, U., Ollinger, K. Lysosomal release of cathepsin D precedes relocation of cytochrome C and loss of mitochondrial transmembrand potential during apoptosis induced by oxidative stress. Free Radic. Biol. Med. 27: 1228-1237, 1999
Roland, G., Rabelink, T.J. Cellular regulation of endothelial nitric oxide synthase. Am. J. Physiol. Renal Physiol. 280: F193-F206, 2001
Rifferl, A., Medina, L.F., Stefani. V., Santos, R.S., Bizani, D., Brandelli, A. In vitro antimacrobial activity of a new series of 1,4-naphthoquinones. Braz. J. Med. Biol. Res. 35: 811-818, 2002
Russell, K.S., Haynes, M.P., Glaser, T.C., Rosneck, J. Sessa, W.C., Bender, J.R. Estrogen stimulates heat shock protein 90 binding to endothelial nitric oxide synthase in human vascular endothelial cells. J. Biol. Chem. 275: 5026-5030, 2000
Shesely, E.G., Maeda, N., Kim, H.S., Desai, K.M., Krege, J.H., Laubach, V.E., Sherman, P.A., Sessa, W.C., Smithies, O. Elevated blood pressures in mice lacking endothelial nitric oxide synthase. Proc. Natl. Acad. Sci. U.S.A 93: 13176-13181,1996
Stephensen, E., Sturve, J., Forlin, L. Effects of redox cycling compounds on glutathione content and activity of glutathione-related enzymes in rainbow trout liver. Comp. Biochem. Physiol. 133: 435-442, 2002
Sweeney, H.L., Yang, Z., Zhi, G., Stull, J.T., Trybus, K.M. Charge replacement near the phosphorylatable serine of the myosin regulatory light chain mimics aspects of phosphorylation. Proc. Natl. Acad. Sci. U.S.A 91: 1490-1494, 1994
Szabo, C. Multiple pathways of peroxynitrite cytotoxicity. Toxicol. Lett. 11: 105-112, 2003
Tikkanen, L., Matsushima, T., Natori, S., Yoshihira, K. Mutagenicity of natural naphthoquinones and benzoquinones in the Salmonella / microsome test. Mutat. Res. 124: 25-34, 1983
Vivar, J.V., Kalyanaraman, B., Martasek, P., Hogg, N., Masters, B.S.S., Karoui, H., Tordo, P., Pritchard, K.A. Superoxide generation by endothelial nitric oxide synthase: The influence of cofactors. Proc. Natl. Acad. Sci. 95:9220-9225, 1998
Vlassara, H., Palace, M.R. Diabetes and advanced glycation endproducts. Journal of internal medicine 251: 87-101, 2002
Wever, R.M.F., Dam, T., Rijn, H.J.M., Groot, F.D., Rabelink, T.J. Tetrahydrobiopterin regulates superoxide and nitric oxide generation by recombinant endothelial nitric oxide synthase. Biochem. Biophys. Res. Commun. 237: 340-344, 1997
Wheatcroft, S.B., Williams, I.L., Shah, A.M., Kearney, H.T. Pathophysiological implications of insulin resistance on vascular endothelial function. Diabet. Med. 20(4): 255-268, 2003
Whorton, A.R., Simonds, D.B., Plantadosi, C.A. Regulation of nitric oxide synthesis by oxygen in vascular endothelial cells. Am. J. Physiol. 272: L1161-L1166, 1997