- 學位論文
iNOS在LPS與H2O2誘發COX-2的角色探討
Inducible nitric oxide synthase (iNOS) is indispensable for lipopolysaccharide- and hydrogen peroxide-induced cyclooxygenase-2 expression in macrophages
摘要
LPS活化巨噬細胞會釋放大量的reactive oxygen species (ROS)來完成在免疫系統的各種細胞功能。Cyclooxygenase-2 (COX-2)和inducible nitric oxide synthase (iNOS)是NF-kB的標的基因，為主要參與此過程的兩個酵素。本篇我們證實巨噬細胞以LPS及H2O2可增加iNOS表現，NO釋出及COX-2表現。以LPS處理巨噬細胞會經Toll-like receptor 4來傳遞訊息，產生ROS及使NF-kB活化。以PDTC (為NF-kB抑制劑)前處理巨噬細胞，雖然可以同步抑制LPS及H2O2所誘導iNOS及COX-2表現，但我們並不能排除LPS，H2O2所誘增的COX-2與NO的釋出有關。確實，1400W (iNOS抑制劑)和ODQ (sGC抑制劑)可以抑制LPS及H2O2所誘導COX-2的表現，暗示參與此訊息傳遞路徑為iNOS→NO→ sGC→cGMP。另外以SNP (NO donor)及 8-Bromo-cGMP (cGMP類似物)處理Raw264.7巨噬細胞，和直接在老鼠腹腔打入SNP及8-Bromo-cGMP所取出的巨噬細胞，皆可證明NO誘導COX-2的表現為一生理現象。為了要更進一步證實我們的假設，我們利用了iNOS-null macrophages。與正常巨噬細胞對照，LPS及H2O2處理iNOS-/-巨噬細胞，我們並不能測得COX-2表現。而另外處理SNP及8-Bromo-cGMP於 iNOS-/-巨噬細胞，則其COX-2表現同正常巨噬細胞均能明顯增加。由上述之結果，我們更能確定LPS與H2O2於巨噬細胞誘增COX-2過程中，iNOS是不可或缺的。
並列摘要
Lipopolysaccharide (LPS)-activated macrophages release large amounts of reactive oxygen species (ROS) to perform a variety of cellular functions in immune system. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), the two well-known nuclear factor-kB (NF-kB) targets, are two of the major enzymes involved in this process. In this study, we demonstrate that LPS and H2O2 increase iNOS expression, nitric oxide (NO) release, and COX-2 expression in macrophages. Following LPS treatment, the signal is transduced into macrophages via Toll-like receptor 4, ROS and leads to NF-kB activation . Though PDTC (a NF-kB inhibitor) can simultaneously abrogate the induction of iNOS and COX-2 in macrophages in response to LPS and H2O2, we can not exclude the possibility that LPS- and H2O2 - mediated COX-2 induction is dependent on the increased release of NO. Indeed, LPS- and H2O2-mediated COX-2 induction could be inhibited by 1400W (an iNOS inhibitor) and ODQ (a sGC inhibitor), suggesting the participation of iNOS→NO→ sGC→cGMP pathway. Consistently, COX-2 can be induced by SNP (a NO donor) and 8-Bromo-cGMP (cGMP analogue) both in murine Raw264.7 macrophages and peritoneal macrophages derived from SNP- and 8-Bromo-cGMP-challenged rats. Further confirmatory evidence derived from studies conducted in iNOS-null macrophages. Compared to wild type macrophages, we fail to detect COX-2 induction in LPS- and H2O2-treated iNOS-/- macrophages. By contrast, augmented expression of COX-2 can be detected in iNOS-/- macrophages following SNP and 8-Bromo-cGMP exposure. With these results, we confirm that iNOS is indispensable in the induction of COX-2 in macrophages.