研究生: |
余宛真 Weng-Jean Yu |
---|---|
論文名稱: |
內毒素處理下黃芩素對誘導型一氧化氮合成酵素與第二型環氧化酵素的影響 The effect of baicalin on inducible nitric oxide synthase and cyclooxygenase-2 expression in lipopolysaccharide-treated macrophage RAW264.7 |
指導教授: |
李銘亮
Li, Ming-Liang |
學位類別: |
碩士 Master |
系所名稱: |
生命科學系 Department of Life Science |
論文出版年: | 2001 |
畢業學年度: | 89 |
語文別: | 中文 |
論文頁數: | 66 |
中文關鍵詞: | 黃芩素 、一氧化氮 、前列腺素E2 、誘導型一氧化氮合成酵素 、第二型環氧化酵素 、腫瘤壞死因子-(alpha) 、細胞株RAW264.7 |
英文關鍵詞: | baicalin, nitric oxide, prostaglandin E2, inducible nitric oxide synthase, cyclooxygenase-2, TNF-(alpha), RAW264.7 cell line |
論文種類: | 學術論文 |
相關次數: | 點閱:236 下載:1 |
分享至: |
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
中文摘要
由中藥黃芩根部萃取出來的黃芩素,在近來研究上具有抗氧化和抗發炎的藥效。許多研究指出,體內過量一氧化氮(nitric oxide, 簡稱NO)、前列腺素E2(prostaglandin PGE2, 簡稱PGE2)、自由基和腫瘤壞死因子-a(tumor necrosis factor-a, 簡稱TNF-a)與敗血性休克的形成有關。而過量NO與PGE2的生成分別是經由誘導型一氧化氮合成酵素(inducible nitric oxide synthase, 簡稱iNOS)與第二型環氧化酵素(cyclooxygenase-2, 簡稱COX-2)所產生。本研究是以內毒素(lipopolysaccharide, 簡稱LPS)刺激細胞株RAW264.7探討黃芩素對於NO、PGE2和TNF-a生成量的影響與iNOS及COX-2的表現。
從我們的結果可知,在以適當濃度LPS(仍可保持良好的細胞活性)刺激下的細胞株RAW264.7,隨著黃芩素處理濃度(10~50 mM)的增高,抑制NO、PGE2和TNF-a生成的效果越好。再者,黃芩素也可以抑制因LPS誘導所產生的iNOS及COX-2的mRNA與蛋白質表現。由結果可推知,黃芩素抑制iNOS及COX-2的mRNA與蛋白質表現的機轉可能是經由抑制TNF-a過量的生成。因此,黃芩素對於治療敗血性休克有很大潛力。另外,以50 mM黃芩素處理細胞株RAW264.7可降低細胞週期中S期和G2/M期的比例,所以黃芩素可能也可當為治療腫瘤的藥劑。
Abstract
It has been demonstrated that baicalin extracted from the root of Huangquin, a Chinese herb medicine has potential antioxidant and anti-inflammatory effect. Many studies have indicated that overproduction of nitric oxide (NO), from inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2) from cyclooxygenase-2 (COX-2), free radical, and tumor necrosis factor-a (TNF-a) is involved in the pathogenesis of septic shock. In this study, we evaluated the effect of baicalin on these mediators and iNOS as well as COX-2 expression in lipopolysaccharide (LPS)-treated RAW264.7 macrophage.
Our results demonstrated that baicalin ( 10~50 mM ) concentration-dependently inhibited LPS-induced NO、TNF-a and PGE2 production in LPS-treated RAW264.7 macrophage without an appreciable cytotoxic effect. Furthermore, baicalin inhibited the expression of LPS-induced iNOS and COX-2 proteins and mRNAs. These results indicated that the inhibition of TNF-a production by baicalin maybe contribute to the suppression of iNOS and COX-2 expressions. Thus, baicalin may be beneficial for treatment of endotoxaemia. Besides, treatment of the RAW264.7 macrophage with 50 mM baicalin decreased the S phase and G2/M phase ratio about 15% in the cell cycle. Thus, baicalin may be also a potential therapeutic drug for tumor therapy.
參考文獻
Anderson, G. D., Hauser, S. D., McGarity, K. L., Bremer, M. E., Isakson, P. C., Gregory, S. A.(1996)Selective inhibition of cyclooxygenase(COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. J. Clin. Invest. 97:2672-2679.
Anggard, E.(1994)Nitric oxide: mediator, murderer, and medicine. Lancet 343:1199-1206.
Baeuerle, P. A. & Baltimore, D.(1988)IkB: A specific inhibitor of NF-kB transcription factor. Science 242: 540-546.
Beasley, D., Cohen, R. A., Levinsky, N. G.(1989)Interleukin-1 inhibits contraction of vascular smooth muscle. J. Clin. Invest. 83:331-335.
Beg, A. A., Finco, I. S., Nantermet, P. V. (1993)Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of IkB-a: a mechanism for NF-kB activation. Mol. Cell. Biol. 13: 3301-3310.
Beutler, B. A., Milsark, I. W., Cerami, A.(1985)Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo. J. Immunol. 135: 3972-3977.
Bredt, D. S., Hwang, P.M., Glatt, C. E., Lowenstein, C., Reed, R. R. and Synder, S. H.(1991)Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 351: 714-718.
Busse, R. & Mulsch, A.(1990)Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells. FEBS Lett. 275: 87-90.
Bustos, M., Coffman, T. M., Saadi, S., Platt, J. L.(1997)Modulation of eicosanoid metabolism in endothelial cells in a xenograft model. Role of cyclooxygenase-2. J. Clin. Invest. 100:1150-1158.
Castrol, L., Rodriguez, M., Radi, R.(1994)Aconitase is readily inactivated by peroxynitrite, but not its precursor, nitric oxide. J. Biol. Chem. 269:29409-29415.
Chen, C. C., Chiu, K. T., Sun, Y. T., Chen, W. C.(1999)Role of the cyclic AMP-protein kinase A pathway in lipopolysaccharide-induced nitric oxide synthase expression in RAW264.7 macrophage. J. Biol. Chem. 207:31559-31564.
Chen, Y. C., Yang, L. L. & Lee, T. J.(2000)Oroxylin A Inhibition of Lipopolysaccharide-Induced iNOS and COX-2 Gene Expression via Suppression of Nuclear Factor-kB Activation. Biochemical Pharmacology 59:1445-1457.
Denizot, F. & Lang, R.(1986)Rapid colorimetric assay for cell growth and survival: modification to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89:271-277.
Eich, R. F., Li, T., Lemon, D. D., Doherty, D. H., Curry, S. R., Aitken, J. F., Mathews, A. J., Johnson, K. A., Smith, R. D., Phillips, G. N. Jr. and Olson, J. S.(1996)Mechanism of NO-induced oxidation of myoglobin and hemoglobin. Biochemistry 35:6976-6983.
Ellis, R. E., Yuan, J. and Horvitz, H. R.(1991)Mechanisms and functions of cell death. Annu. Rev. Cell. Biol. 7:663-698.
Fletcher, J. R., Collins, J. N., Graves, E. D., et al.(1993) Tumor necrosis factor-induced mortality is reversed with cyclooxygenase inhibition. Ann. Surg. 217:668.
Gardiner, S. M., Compton, A. M. & Bennett, T.(1990)Control of regulatory blood flow by endothelium-derived nitric oxide. Hypertension 15:485-492.
Gibbs, Jackson B.(2000)Mechanism-based target identification and drug discovery in cancer research. Science 287:1969-1973.
Grathwaite, J., Charlas, S. L. & Chess-Williams, R.(1988)Endothelium-derived relaxating factor released on activation of NMDA receptors suggests a role as intercellular messenger in the brain. Nature 336: 385-388.
Green, L.M., Reade, J. L., & Ware, C.F.(1984)Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines. J. Immunol. Methods 70:257-268.
Greenberg, S. S., Xie, J., Zartrain, J. M., et al(1995)Hydroxycobalamin(vitamin B12) prevents and reverse endotoxin-induced hypotension and mortality in rodents: Role of nitric oxide. J. Pharmacol. Exp. Ther. 273:257-265.
Griffith, O. W. & Stuehr, D. J.(1995)Nitric oxide synthase: properties and catalytic mechanism. Annu. Rev. Physiol. 57:707-736.
Groeneveld, A. B. J., Bronsveld, W., Thijp, L. G.(1986)Hemodynamic determinants of mortality in human septic shock. Surgery 99: 140-152.
Hathaway, D. R., Konicki, M. V., Coolican, S. A.(1985)Phosphorylation of myosine light chain kinase from vascular smooth muscle by cAMP and cGMP dependent protein kinase. J. Mol. Cell. Cardiol. 17:841-850.
Hellstrom, W. J. G., Bell, M., Wang, R., Sikka, S. C.(1994)Effect of sodium nitroprusside on sperm motility, viability, and lipid peroxidation. Fertil. Steril. 61: 1117-1122.
Hibbs, J. J., Taintor, R., Vavrin, Z., Rachlin, E. (1988) Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. Biophys. Res. Commun. 157:87-94.
Hisakawa, N., Nishiya, K., Tahara, K., Matsumori, A., Hashimoto, K., (1998) Down regulation by iron of prostaglandin E2 production by human synovial fibroblast. Annal. Rheumatic Disease 57: 742-746.
Huang, Q., Yuan, Y.(1997)Interaction of PKC and NOS in signal transduction of microvascular hyperpermeability. Am. J. Physiol. 273:2442-2451.
Ikemoto, S., Sugimyra, K., Yoshida, N., Yasumoto, R., Wada, S.,Yamamoto, K., Kishimoto, T.(2000)Antitumor effects of Scutellariae radix and its components baicalin, baicalein and wogonin on bladder cancer cell line. Urology 55:951-955.
Inmaculada-Posadas, M., Carmen, T., Guillen, I. M., Luisa, F., et al.(2000)Co-regulation between cyclooxygenase-2 and inducible nitric oxide synthase expression in the time-course of murine inflammation. Naunyn-Schmiedeberg’s Arch. Pharmacol. 361:98-106.
Jacobs, E. R., Soulsby, M. E., Bone, R. C., et al.(1982)Ibuprofen in canine endotoxin shock. J. Clin. Invest. 70:536.
Kasai, K., Hattori, Y., Banba, N., et al.(1997)Induction of tetrahydrobiopterin synthesis in rat cardiac myocytes: Impact on cytokine-induced NO generation. AM J Physiol. 273:665-672.
Lo, C., Cryer, H., Fu, M., Lo, F.,(1998)Regulation of macrophage eicosanoid generation is depend on nuclear factor kappa B. J. Trauma 45:19-24.
Lowenstein, C. J., Alley, E. W., Raval, P., Snowman, A. M., Synder, S. H., Russell, S. W. & Murphy, W. J.(1993)Macrophage nitric oxide synthase gene:two upstream regions mediate induction by interferon g and lipopolysaccharide. Proc. Natl. Acad. Sci. USA 90:9730-9734.
MacMicking, J. D., Nathan, C., Home, G., et al.(1995)Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81:641-650.
MacMicking, J., Xie, Q. & Nathan, C.(1997)Nitric oxide and macrophage function. Annu. Rev. Immunol. 15:323-350.
Meckenna, T. M.(1990)Prolonged exposure of rat aorta to low levels of endotoxin in vitro results in impaired contractility. J. Clin. Invest. 86:160-168.
Mellion, B. T., Ignarro, L. J., Ohlstein, E. H., Pontecorvo, E. G., Hyman, A. L. and Kadowitz, P. J.(1981)Evidence for the inhibitory role of guanosine 3’,5’-monophosphate in ADP-induced human platelet aggregation in presence of nitric oxide and released vasodilators. Blood 57:946.
Meyer, M., Schreck, R. & Baeuerle, P. A.(1993)H2O2 and antioxidants have opposite effects on activation of NF-kB and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 12: 22005-22015.
Mitaka, C., Hirata, Y., Ichikawa, K., Uchide, T., Yokoyama, K., Nagura, T., Tsunoda, Y., Amaha, K.(1995)Effect of nitric oxide synthase inhibitor on hemodynamic change and O2 delivery in septic dogs. Am. J. Physiol. 268: 2017-2023.
Mohamed, F., Monge, J. C., Gordon, A., Cernacek, P., Blais, D., Stewart, D. J. ( 1995 ) Lack of role of nitric oxide (NO) in the selective destabilization of endothelial NO synthase mRNA by tumor necrosis factor-a. Arterioscler. Thromb. Vasc. Biol. 15:52-57.
Moncada, S., Palmer, R. M. & Higgs, A.(1991)Nitric oxide: physiology, Pathophysiology, and pharmrcology. Pharmacol. Rev. 43:109-142.
Nathan, C. & Xie, Q.(1994) Regulation of biosynthesis of nitric oxide. J. Biol. Chem. 269: 13725-13728.
Nishio E., Watanabe, Y.(1997)Role of the lipooxygenase pathway in phenylephrine-induced vascular smooth muscle cell proliferation and migration. Eur. J. Pharmacol. 336: 267-273.
Nomura, Y. & Kitamura, Y.(1993)Inducible nitric oxide synthase in glial cells. Neurosci. Res. 18:103-107.
Oddis, C. V., Finkel, M. S.(1996)NF-kB, and GTP cyclohydrolase regulate cytokine-induced nitric oxide production by cardiac myocytes. Am. J. Physiol. 270:1568-1574.
Okamoto, H., Ito, O., Roman, R. J., and Hudetz, A. G.(1998)Role of inducible nitric oxide synthase and cyclooxygenase-2 in endotoxin-induceed cerebral hyperemia. Stroke 29:1209-1218.
Palmer, R. M. J., Ashton, D. A. & Moncada, S.(1988)Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature(Lond.)333:664-666.
Parratt, J. R.(1973)Myocardial and circulatory effects of E. coli endotoxin; modification of responses to catecholamines. Br. J. Pharmacol. 47:12-25.
Pascual, D. W., Pascual, V. H., Bost, K. L., McGhee, R. J. and Oparil, S.(1993)Nitric oxide mediates immune dysfunction in the spontaneously hypertensive rat. Hypertension 21: 185-194.
Rocca, B., Spain, L. M., Pure, E., Langenbach, R., Patrono, C., FitzGerald, G. A.,(1999)Distinct roles of prostaglandin H synthase 1 and 2 in T-cell development. J. Clin. Invest. 103: 1469-1477.
Schmidt, H. H., Nau, H., Wittfont, W., Gerlach, J., Prescher, K. E., Klein, M. M., Niroomand, F., and Bohme, E.(1988)Arginine is a physiological precursor of endothelium-derived nitric oxide. Eur. J. Pharmacol. 154:213-216.
Schmidt, H. H. H. W., Lohmann, S. M., Walter, U.(1993)The nitric oxide and cGMP signal transduction system: Regulation and mechanism of action. Biochem. Biophys. Acta. 1178:153-175.
Schraufstatter, I. U., Hinshaw, D. B., Hyslop, P. A., et al.(1986)Oxidant injury of cells: DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide. J. Clin. Invest. 77: 1312-1320.
Schreck, R. & Baeuerle, P. A.(1994)Assessing oxygen radicals as mediators in activation of inducible eukaryotic transcription factor NF-kB. Meth. Enzy. 234:151-163.
Shi, H., Zhao B., Xin, W.(1995)Scavenging effects of baicalin on free radicals and its protection on erythrocyte membrane from free radical injury. Biochem. Mole. Biol. Internal. 35: 981-984.
Suffredini, A. F., Fromm, R. E., Parker, M. M.(1989)The cardiovascular response of normal humans to the administration of endotoxin. N. Engl. J. Med. 321:280-287.
Szabo, C., Saunders, C., O'Connor, M., et al.(1997)Peroxynitrite causes energy depletion and increases permeability via activation of poly( ADP-ribose ) synthetase in pulmonary epithelial cells. Am. J. Respir. Cell Mol. Biol. 16:105-109.
Szabo1, C., Zingarelli, B., O'Connor, M., et al. ( 1996 ) DNA breakage, activation of poly-ADP ribosyl synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite. Proc. Natl. Acad. Sci. USA 93:1753-1758.
Szabo2, C., Zingarelli, B., Salzman, A. L.(1996)Role of poly-ADP ribosyltransferase activation in the vascular contractile and energetic failure elicited by exogenous and endogenous nitric oxide and peroxynitrite. Circ. Res. 78: 1051-1063.
Tracey, K. J., Beutler, B., Lowry, S. F., Merryweather, J., Wolpe, S., Milsark, I. W., Hairiri, R. J., Fahey, T. J., Zentella, A., Albert, J. D., Shires, G. T., Cerami, A.(1986)Shock and tissue injury induced by recombinant human cachectin. Science 234: 470-473.
Tracey, K. J., Fong, Y., Hesse, D. G.(1987)Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330: 662-664.
Walton, L. J., Franklin, I. J., Bayston, T., Brown, L. C., Greenhalgh, R. M., Taylor, G. W., Powell, J. T.(1999)Inhibition of prostaglandin E2 synthesis in abdominal aortic aneurysms:implication for smooth muscle cell viability, inflammatiory process, and the expansion of abdominal aortic aneurysms. Circulation 100:48-54.
Xiao, J. & Pang, P. K. T.(1994)Dose a general alteration in nitric oxide synthesis system occur in spontaneously hypertensive rats? Am. J. Physiol. 266:272-278.
Xie, Q., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T. & Nathan C.(1992)Clonig and characterization of inducible nitric oxide synthase from mouse macrophage. Science 256:225-228.
Xie, Q., Whisnant, R.&Nathan, C.(1993)Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon g and bacterial lipopolysaccharide. J. Exp. Med. 177:1779-1784.
You, K. M., Jong, H. G., Kim, H. P.(1999)Inhibition of cyclooxygenase / lipoxygenase from human platelets by polyhydroxylated / methoxylated flavonoids isolated from medicine. Arch. Pharmacal Research 22:18-24.
新編中藥大辭典(下) 新文豐出版公司印行 / 中華民國七十四年再版 第132頁至第137頁。