AGEs caused a dose-dependent increase of nitrite accumulation in C6 glioma cells

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過度糖化最終產物刺激C6 神經膠瘤細胞表現誘導型一氧化氮合成酵 素之訊息傳遞路徑

Signal Transduction of Advanced Glycosylation End

Products(AGEs) Stimulated Inducible-Nitric Oxide Synthase Expression in C6 Glioma Cells

中文摘要

在阿茲罕默氏症（Alzheimer’s disease）及其他許多退行性神經病變中，誘導型一氧化氮合成酵素（inducible nitric oxide synthase，簡稱 iNOS）的活性扮演著重要的角色，而iNOS 的蛋白表現會受到 mitogen-activated protein

kinase（MAPK）的調控。我們在實驗中，以過度糖化最終產物（BSA-AGEs）

誘導C6 神經膠瘤細胞（C6 glioma cells）iNOS 的蛋白表現，發現 p38 MAPK 在其訊息傳遞過程扮演相當重要的角色。利用BSA-AGEs 刺激 C6 glioma cells，不論是隨著劑量的增加或是反應時間的增加，iNOS 的蛋白表現以及一氧化氮

（NO）的產生都會隨之增加，而這種現象可以被基因轉錄抑制劑 actinomycin D，基因轉譯抑制劑 cyclohexamide，和 iNOS 抑制劑 Nw-nitro-L-arginine methyl easter（L-NAME）所抑制，由此可以推測 iNOS 的蛋白表現是由基因表現而來，

而NO 的增加則是由於 iNOS 的正調節作用（up-regulation）所致。另外，預先以 tyrosine kinase 抑制劑（genistein 和 tyrphostin）、Ras-farnesyl transferase 抑制劑- II（FPT-II）、p38 MAPK 抑制劑（SB203580）及 MEK 抑制劑（PD98059）等處理C6 glioma cells，都可以抑制由 BSA-AGEs 所刺激的 iNOS 表現及 NO 產生。 BSA-AGEs 可以促使 C6 神經膠瘤細胞 p38 MAPK 的活性增加，而此一現象會被 genistein（20mM）、tyrphostin（30mM）、FPT inhibitor-II（20mM）和

SB203580（10mM）所抑制，另外，BSA-AGEs 也可以促使 ERK 的活性增加，

相同地，也會被genistein（30mM）、FPT inhibitor-II（20mM）和

PD98059（10mM）所抑制。綜合以上結果，我們的研究顯示，BSA-AGEs 在 C6 glioma cells 中的訊息傳遞路徑是經由活化 tyrosine kinase 和 Ras，導致 p38 MAPK 及 ERK 的活化，而進一步的促使 iNOS 表現而產生 NO。

英文摘要

Inducible nitric oxide synthase (iNOS) activity plays important roles in Alzheimer’s disease and other neurodegeneration diseases. The mitogen-activated protein kinase (MAPK) pathway is believed to function as an important mediator of iNOS

expression. In the present study, we investigated the role of the p38 MAPK signaling pathway in advanced glycosylation end products (AGEs)-induced iNOS expression in C6 glioma cells. AGEs caused a dose-dependent increase of nitrite accumulation in C6 glioma cells. The AGEs-stimulated nitrite production from C6 glioma cells was inhibited by actinomycin D, cycloheximide, and the NO synthase inhibitor, Nw-nitro-

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L-arginine methyl ester (l-NAME), suggesting that the increase of AGEs-induced nitrite release is due to iNOS up-regulation. Consistently, treatment of C6 glioma cells with AGEs induced iNOS protein expression. The tyrosine kinase inhibitor (genistein

& tyrphostin), the Ras-farnesyl transferase inhibitor (FPT inhibitor-II), the p38 MAPK inhibitor (SB203580), or the MEK inhibitor (PD98059) suppressed AGEs- induced iNOS expression and nitrite release from C6 glioma cells. AGEs activated p38 MAPK in C6 glioma cells, and this effect was blocked by genisteine (20 mM), tyrphostin (30 mM), FPT inhibitor-II (20 mM), and SB203580 (10 mM). AGEs also activated ERK in C6 glioma cells, and this effect was blocked by genisteine (30 mM), FPT inhibitor-II (30 mM), and PD98059 (10 mM). Taken together, our data suggest that AGEs may activate the pathways of tyrosine kinase and Ras to induce p38 MAPK and ERK activation, which in turn induces iNOS expression and NO production in C6 glioma cells.