肽聚醣經由 c-Src 和 NADPH 氧化酶之路徑誘導 RAW 264.7 巨噬細
胞環氧化酶-2 的表現
Involvement of c-Src and NADPH oxidase in Peptidoglycan-Induced Cyclooxygenase-2 Expression in RAW 264.7 Macrophages
中文摘要
肽聚醣 (peptidoglycan；PGN)，革蘭氏陽性菌金黃葡萄球菌細胞壁成分；它會活 化宿主的免疫系統，並誘導發炎物質的釋放。本篇論文主要是研究在 RAW 264.7 巨噬細胞中，cell Src (c-Src) 和 nicotinamide adenine dinucleotide
phosphate-dependent 氧化酶 (NADPH oxidase) 在 PGN 誘導 COX-2 的表現中 所扮演的角色。由 PGN 誘導 COX-2 的表現會被 c-Src dominant-negative mutant (c-SrcDN)、NADPH oxidase 抑制劑 diphenyleneiodonium chloride (DPI) 和抗氧化劑 glutathione (GSH) 及 N-acetylcysteine (NAC) 所抑制。PGN 會增加 c-Src 的磷酸化以及活化。PGN 會時間依賴性地誘導 NADPH oxidase 次單元 p47phox 由細胞質轉位到細胞膜上，此現象會被 c-SrcDN 所抑制。PGN 也會時 間依賴性的增加 reactive oxygen species (ROS) 的產生，此現象會被 DPI、GSH 和 NAC 所抑制。而 PGN 所誘導的 apoptosis signal-regulating kinase 1 (ASK1) 的活化可以被 c-SrcDN、DPI、GSH 和 NAC 所抑制。此外，PGN 誘導的 c-Jun NH2-terminal kinase 1/2 (JNK1/2) 的活化也會被 c-SrcDN、GSH 和 NAC 所抑 制。進一步的研究顯示，PGN 會誘導 Toll-like receptor 2 (TLR2)、c-Src 以及 p47phox 複合體的產生。我們的結果證實在 RAW 264.7 巨噬細胞中，PGN 會 活化 c-Src/NADPH oxdiase 訊號傳遞，進
而促使 ROS 的釋放，並活化下游 ASK1 與 JNK，最終導致 COX-2 的表現。
英文摘要
Peptidoglycan (PGN), the Gram-positive bacterium Staphylococcus aureus cell wall component, activates the immune system of host and induces release of inflammatory mediators. In this study, we investigated the roles of that cell c-Src (c-Src) and
NADPH oxidase in PGN-induced cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophage. The PGN-induced COX-2 expression was attenuated by the dominant negative mutant of c-Src (c-SrcDN), the NADPH oxidase inhibitor
diphenyleneiodonium chloride (DPI), and the ROS scavenger N-acetylcysteine (NAC) and glutathione (GSH). Treatment of cells with PGN caused an increase in c-Src phosphorylation and c-Src activity. PGN caused a time-dependent increase in the translocation of the p47phox subunit of NADPH oxidase from the cytosol to the membrane fractions, which was attenuated by c-SrcDN. PGN caused a
time-dependent increase in reactive oxygen species (ROS) formation, this effect was attenuated by DPI, GSH, and NAC. The PGN-induced apoptosis signal-regulating kinase 1 (ASK1) activation were inhibited by c-SrcDN, DPI, NAC, and GSH.
Furthermore, the c-Jun NH2-terminal kinase (JNK) activation caused by PGN was inhibited by c-SrcDN, GSH, and NAC. Further studies revealed that PGN induced TLR2, c-Src, and p47phox complex formation. Our results demonstrated that PGN activates the c-Src/NADPH oxdiase pathway to induce ROS release, which in turn initiates the activations of ASK1 and JNK, and ultimately induces COX-2 expression in RAW 264.7 macrophages.
