Nuclear Factor-kappa B在過度醣化最終產物誘導RAW 264.7巨噬細胞表現一氧化氮合成酶中所扮演的角色

Involvement of Nuclear Factor-kappa B in Advanced Glycosylation End Products-Induced Inducible Nitric Oxide Synthase Expression in RAW 264.7 Macrophages

黃昭銘(ChaoMing Huang)

指導教授 ： 李宏謨

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2001年)

摘要

過度醣化最終產物(Advanced glycosylation end products, AGEs )已被證實與糖尿病併發症的病理有關。本篇論文主要是探討在RAW 264.7巨噬細胞中，過度醣化最終產物在誘導型一氧化氮合成酶(inducible nitric oxide synthase, iNOS)表現的影響。利用過度醣化最終產物處理RAW 264.7巨噬細胞後，一氧化氮的產生和誘導型一氧化氮合成酶的表現會以劑量及時間相關性增加。Nuclear factor-kappa B (NF-kB)之抑制劑pyrrolidone dithiocarbamate (PDTC)會抑制過度醣化最終產物所引起一氧化氮的產生和誘導型一氧化氮合成酶的表現。若以過度醣化最終產物處理RAW 264.7巨噬細胞30分鐘會引起NF-kB由細胞質轉位到細胞核內。在electrophoretic mobility shift assay (EMSA)的結果中，過度醣化最終產物也會增加NF-kB結合基因的能力。而Tyrosine kinase之抑制劑genistein、phosphatidylinositol-3-kinase (PI-3K)之抑制劑LY 294002、phosphatidylcholine-phospholipase C (PC-PLC)之抑制劑D 609、protein kinase C (PKC)之抑制劑Ro 31-8220和p38 mitogen-activated protein kinase (MAPK)之抑制劑SB 203580會抑制過度醣化最終產物所引起一氧化氮的產生和誘導型一氧化氮合成酶的表現，表示tyrosine kinase、PI-3K、PC-PLC、PKC、p38 MAPK和NF-kB參與此訊號傳遞路徑中。而NF-kB的轉位和結合基因的能力也會被genistein、LY 294002、D 609、Ro 31-8220和SB 203580所抑制。綜合以上的結果可知過度醣化最終產物刺激RAW 264.7巨噬細胞的一氧化氮產生和誘導型一氧化氮合成酶表現是經由NF-kB的活化，而活化NF-kB的上游訊號傳遞路徑可能有tyrosine kinase、PI-3K、PC-PLC、PKC、p38 MAPK的參與。

關鍵字

過度醣化蛋白最終產物 ； 誘導型一氧化氮合成酶轉錄因子NF-kB ； RAW 264.7巨噬細胞

並列摘要

Advanced glycosylation end products (AGEs) have been implicated in the pathogenesis of diabetic complications. In this study, the effect of AGEs on inducible nitric oxide synthase (iNOS) expression in RAW 264.7 macrophages has been investigated. Treatment of RAW 264.7 macrophages with AGEs caused a dose- and time-dependent increase of nitric oxide (NO) production and iNOS expression. Treatment of RAW 264.7 macrophages with AGEs for 30 min stimulated the translocation of NF-kB from cytosol to nucleus. Electrophoretic mobility shift assay revealed that the NF-kB DNA-protein binding activity was enhanced by AGEs. AGEs-induced NO production and iNOS expression were blocked by the tyrosine kinase inhibitor, genistein, the phosphatidylinositol-3-kinase (PI-3K) inhibitor, LY 294002, the phosphatidylcholine-phospholipase C (PC-PLC) inhibitor, D 609, the protein kinase C (PKC) inhibitor, Ro 31-8220, the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB 203580, and the nuclear factor-kappa B (NF-kB) inhibitor, pyrrolidone dithiocarbamate (PDTC), suggesting that tyrosine kinase, PI-3K, PC-PLC, PKC, p38 MAPK and NF-kB are involved in this signaling event. AGEs-stimulated translocation and DNA binding activity of NF-kB were inhibited by genistein, LY 294002, D 609, Ro 31-8820, SB 203580 and PDTC. In conclusion, AGEs might activate NF-kB via an upstream signaling cascade which composed of tyrosine kinase, PI-3K, PC-PLC, PKC, and p38 MAPK, resulting the induction of iNOS protein expression and NO release in RAW 264.7 macrophages.

並列關鍵字

advanced glycosylation end products ； inducible nitric oxide synthase ； nuclear factor-kappa B ； RAW 264.7 macrophages

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