Dipyridamole抑制RAW 264.7巨噬細胞中脂多醣體誘導的ㄧ氧化氮合成酶與環氧酵素-2的表現

Dipyridamole Inhibits Lipopolysaccharide-Induced Inducible Nitric Synthase and Cyclooxygenase-2 Expression in RAW 264.7 Cells

高遠忠(Yuan-Chung Kao)

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(2004年)

摘要

Dipyridamole是一個核苷運送的抑制劑，也是一個非選擇性的phosphodiesterase的抑制劑，因此能夠藉由抑制phosphodiesterase的機制來增加細胞內cAMP以及cGMP的濃度。第四型的phosphodiesterase抑制劑已經在許多實驗中被證實具有抗發炎的功能，本研究所要探討的主題就是，在RAW 264.7巨噬細胞中Dipyridamole是否可以抑制Lipopolysaccharide (LPS) 誘導的iNOS以及COX-2的表現。以LPS處理RAW 264.7巨噬細胞會造成iNOS以及COX-2以劑量依存性及時間依存性表現。若以Dipyridamole前處理細胞則可以阻斷LPS所誘導的iNOS及COX-2表現。藉由抑制I?B phosphorylation、degradation、p65 NF-?B translocation以及reporter gene的轉錄作用的方式來證明Dipyridamole會抑制NF-?B路徑的活化。另外，Dipyridamole也可以抑制LPS在RAW 264.7細胞中所造成的p38 MAPK以及IKK-?的活化。若進一步以p38 MAPK的抑制劑SB203580前處理細胞，則能抑制LPS誘導的iNOS表現以及IKK-?活化，所以LPS是先活化了p38 MAPK，再活化NF-?B的訊息傳遞路徑。另外，Dipyridamole能夠刺激mitogen-activated protein kinase phosphatase 1 (MKP-1) 的磷酸化及活化而使得p38 MAPK去磷酸化及去活化而失去功能。總而言之，本研究證明在RAW 264.7巨噬細胞中，Dipyridamole會先藉由活化MKP-1的方式使得p38 MAPK去磷酸化而失去功能。然而p38 MAPK去活化後，接著就會抑制IKK-?的活化以及後續由NF-?B所調控的訊息傳遞路徑，因而抑制LPS所誘導的iNOS及COX-2表現。本研究的結果支持 dipyridamole具抗發炎作用的假說。

關鍵字

誘導型ㄧ氧化氮合成酶環氧酵素 ； 脂多醣體

並列摘要

Dipyridamole is a nucleoside transport inhibitor and a non-selective phosphodiesterase inhibitor that increases intracellular level of cAMP and cGMP through phosphodiesterase inhibition. Type 4 phosphodiesterase has been demonstrated to have anti-inflammatory effects in many experimental systems. This study investigates whether dipyridamole inhibits lipopolysaccharide (LPS)-induced inducible nitric oxide (iNOS) and cyclooxygenase (COX-2) expression in RAW 264.7 macrophages. Treatment of RAW 264.7 macrophages with LPS caused dose- and time-dependent increases in iNOS and COX-2 expression. Treatment of cells with dipyridamole blocked the LPS-induced iNOS and COX-2 expression. Dipyridamole inhibited NF-?B activation as demonstrated by inhibition of I?B phosphorylation, I?B degradation, p65 NF-?B translocation and the transcription of reporter gene. Dipyridamole also inhibited LPS-stimulated p38 MAPK and IKK-? activities in RAW 264.7 cells. A p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, inhibited LPS-stimulated iNOS expression and IKK-? activation, suggesting LPS may activate NF-?B signaling pathway via upstream p38 MAPK activation. Furthermore, dipyridamole stimulated a transient activation of mitogen-activated protein kinase phosphatase 1 (MKP-1), a potent inhibitor of p38 MAPK. Taken together, these data suggest that dipyridamole exerts anti-inflammatory effect via activation of MKP-1, which dephosphorylates and inactivates p38 MAPK. Inactivation of p38 MAPK in turn inhibits IKK-? activation and subsequent NF-?B signaling pathway that mediates LPS-induced iNOS and COX-2 expression in RAW 264.7 cells. These results support the notion that dipyridamole may have anti-inflammatory effects.

並列關鍵字

inducible nitric oxide synthase ； cyclooxygenase ； lipopolysaccharide

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