奎寧刺激C6神經膠瘤細胞表現誘導型一氧化氮合成酵素之訊號傳遞路徑

中文摘要(第一部分) 論文名稱：奎寧刺激C6神經膠瘤細胞表現誘導型一氧化氮合成酵素之訊號傳遞路徑研究所名稱：臺北醫學大學生物醫學技術研究所研究生姓名：張博喬畢業時間：九十學年度第二學期指導教授：李宏謨博士臺北醫學大學生物醫學技術研究所所長兼教授奎寧(chloroquine)是一種aminoquinoline類的藥物，常被用來治療瘧疾與類風濕性關節炎方面的疾病。奎寧(chloroquine)能誘導許多細胞如大鼠、豬與人類的細胞，誘導型一氧化氮合成酵素的表現。然而，奎寧(chloroquine)調控誘導型一氧化氮合成酵素 (inducible nitric oxide synthase) 表現的機制並未完全了解。本研究中，我們主要探討在C6神經膠瘤細胞(C6 glioma cells)，奎寧(chloroquine)造成誘導型一氧化氮合成酵素表現的訊號傳遞路徑。以奎寧(chloroquine)處理之C6 glioma cells，iNOS的表現與一氧化氮(NO)的產生都有劑量依存性與時間依存性增加，而這些現象無法被氧化還原的抑制劑所抑制；由此可以推斷，chloroquine所引起的iNOS表現和NO的產生與氧化壓力並無相關。預先給予tyrosine kinase的抑制劑(genistein)、p38 MAPK的抑制劑(SB203580)和Protein kinase C的抑制劑(Ro31-8220)處理細胞後，chloroquine所誘導的 iNOS表現和NO生成都受到抑制。另外，chloroquine可以增加p38 MAPK的活性，而此效應可被tyrosine kinase的抑制劑(genistein)、p38 MAPK的抑制劑(SB203580)和Protein kinase C的抑制劑(Ro31-8220)所抑制。另外，LPS亦能引起iNOS的表現和NO的生成，當同時給予LPS及chloroquine時，C6神經膠瘤細胞iNOS的表現和NO的生成均有增強的現象，但也造成細胞的凋亡。綜合以上的結果，chloroquine會藉由tyrosine kinase與PKC的訊息傳遞路徑去調控p38 MAPK的活性，誘導iNOS的表現及一氧化氮的生成，由於被LPS活化的C6神經膠瘤細胞在chloroquine 的存在下會導致C6神經膠瘤細胞的凋亡。因此，很可能chloroquine所導致的iNOS表現及NO的生成是動物用來使過度活化的微膠質細胞凋亡的機制。中文摘要(第二部分) 論文名稱：過度糖化最終產物刺激大鼠環間膜細胞表現誘導型一氧化氮合成酵素之訊號傳遞路徑: p38 MAPK參與在其中研究所名稱：臺北醫學大學生物醫學技術研究所研究生姓名：張博喬畢業時間：九十學年度第二學期指導教授：李宏謨博士臺北醫學大學生物醫學技術研究所所長兼教授引起糖尿病併發症的原因，可能與蛋白質長期處於高糖的環境下，蛋白質慢慢變成不可還原性的過度糖化的最終產物(簡稱AGEs)有關。AGEs在組織的堆積也經證實與糖尿病腎臟併發症有關；在這篇論文中，發現AGEs對於大鼠腎臟環間膜細胞中iNOS的表現與NO的生成有劑量-與時間-依存性的關係。給予tyrosine kinase的抑制劑(Genistein)可以抑制iNOS的表現及NO生成。除此之外， p38 MAPK的抑制劑(SB203580)、NF-kB的抑制劑(PDTC)、Protein kinase C的抑制劑(Ro31-8220)及Protein kinase A的抑制劑(KT5720)可以抑制AGEs刺激的 iNOS表現與NO生成作用；因此，tyrosine kinase的磷酸化、NF-kB的轉位、PKC及PKA的活化，與p38 MAPK的活化，皆參與在AGEs誘導iNOS表現的訊息傳遞路徑。另外，AGEs可以活化p38 MAPK，而此效應會受到tyrosine kinase的抑制劑(Genistein)、p38 MAPK的抑制劑(SB203580)、NF-kB的抑制劑(PDTC)及Protein kinase C的抑制劑(Ro31-8220)所抑制；因此，AGEs所調控的iNOS表現與NO的生成，可能包括了tyrosine kinase的磷酸化、NF-kB的轉位、PKC、PKA及p38 MAPK的活化。

關鍵字

誘導型一氧化氮合成酵素；奎寧； c6神經膠瘤細胞； p38 MAPK

並列摘要

Abstract (part I) Title of Thesis：Chloroquine Induces the Expression of Inducible Nitric Oxide Synthase in C6 Glioma Cells Author：Po-Chiao Chang Thesis advised by：Horng-Mo Lee Ph.D. Chloroquine, a well-known lysosomotropic agent, has been used for treatment of malaria and rheumatologic disorders. However, therapeutic doses of chloroquine are known to cause behavioral side effects. In the present study, we investigated the investigation that whether chloroquine stimulated iNOS expression and NO synthesis in C6 glioma cells. Chloroquine caused dose- and time-dependent increases of iNOS protein expression and NO production in C6 glioma cells. The induction was not affected by L-buthionine-[S, R]-sulfoximine (BSO), gamma-glutamylcysteine synthetase inhibitor, or by L-nitro-acetyl-cysteine (L-NAC), a known glutathione precursor, suggesting that the chloroquine-induced iNOS expression is not due to reactive oxygen species. A tyrosine kinase inhibitor (genistein), a protein kinase C inhibitor (Ro31-8220), or a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) suppressed chloroquine-induced iNOS expression and nitrite release from C6 glioma cells. Chloroquine activated p38 MAPK in C6 glioma cells, and this effect was blocked by genistein (20 mM), Ro31-8220 (3 mM) and SB203580 (10 mM). Incubation of lipopolysacharide (LPS)-stimulated cells with chloroquine at non-toxic concentration (10-100?mM) for 48 h increased iNOS expression, and led to a significant loss of adherent cells. The induction of DNA fragmentation in floating cells indicates that the C6 glioma cells were undergoing apoptosis. Taken together, our data suggest that chloroquine may activate the pathway of tyrosine kinase to induce p38 MAPK activation, which in turn induces iNOS expression and NO production. Our work support the assumption that NO may serve as a mediator to eliminate the over-activated microglial cells by apoptosis. Key Words: iNOS , Chloroquine, C6 glioma cells, p38 MAP kinase. Abstract (part II) Title of Thesis：Advanced Glycosylation End Product Induces the Expression of Inducible Nitric Oxide Synthase in Rat Mesangial Cells: Involvement of p38 MAP Kinase Dependent Mechanisms Author：Po-Chiao Chang Thesis advised by：Horng-Mo Lee Ph.D. Non-enzymatic reaction of protein and carbohydrate produce a series of brown fluorescent advanced glycosylation end products (AGEs), AGEs accumulation in tissue have been implicated in diabetic related complications including diabetic nephropathy. Nitric oxide is a crucial mediator of several forms of glomerular inflammation. In the present study, we investigated whether AGEs regulate iNOS expression and nitrite release in rat mesangial cells. AGEs caused a dose-dependent increase of nitrite accumulation in rat mesangial cells. Consistently, treatment of rat mesangial cells with AGEs stimulated the inducible nitric oxide synthase (iNOS) protein expression. The AGEs-stimulated iNOS expression and NO production from rat mesangial cells was inhibited by a tyrosine kinase inhibitor (genistein), a NF-kB inhibitor (PDTC), a p38 MAPK inhibitor (SB203580), a protein kinase C inhibitor (Ro31-8220)，or a protein kinase A inhibitor (KT5720), suppressed AGEs-induced iNOS expression and nitrite release from rat mesangial cells. AGEs activate p38 MAP kinase in rat mesangial cells and this effect was blocked by genisteine (20mM), PDTC (25mM), Ro31-8220 (3mM) and SB203580 (10mM). Taken together, our data suggest that p38 MAP kinase is involved in AGEs induced iNOS expression and NO production in rat mesangial cells. Key Words: AGEs, iNOS, rat mesangial cells, p38 MAP kinase

並列關鍵字

iNOS ； chloroquine ； C6 glioma cells ； p38 MAP kinase

參考文獻

參考文獻(part I) Barna M., Komatsu T., and Reiss C.S. (1996) Activation of type III nitric oxide synthase in astrocytes following a neurotropic viral infection. Virology 223: 331-343

Buszman E., Kopera M., and Wilczok T. (1984) Electron spin resonance studies of chloroquine-melanin complexes. Biochem. Pharmacol. 33: 7-11

Buzanska L., Zablocka B., Dybel A., Domanska-Janik K., and Albrecht J. (2000) Delayed induction of apoptosis by ammonia in C6 glioma. Neurochem. Int. 37: 287-297

Chen F., Lu Y., Kuhn D.C., Maki M., Shi X., Sun S.C., and Demers L.M. (1997) Calpain contributes to silica-induced IkB-a degradation and nuclear factor-kappa B activation. Arch. Biochem. Biophys. 342: 383-388

Denhardt D.T. (1996) Signal transducing phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signaling. Biochem. J. 318: 89-106

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奎寧刺激C6神經膠瘤細胞表現誘導型一氧化氮合成酵素之訊號傳遞路徑

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