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Cyclooxygenase-2表現在脂臺口酸刺激巨噬細胞一氧化氮生成之路徑探討

Cyclooxygenase-2表現在脂臺口酸刺激巨噬細胞一氧化氮生成之路徑探討.

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Cyclooxygenase-2表現在脂臺口酸刺激巨噬細胞一氧化氮生成之路徑探討

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  1. Cyclooxygenase-2表現在脂臺口酸刺激巨噬細胞一氧化氮生成之路徑探討Cyclooxygenase-2表現在脂臺口酸刺激巨噬細胞一氧化氮生成之路徑探討 • 本論文主要探討lipoteichoic acid (LTA) 刺激RAW 264.7巨噬細胞cyclooxygenase-2 (COX-2) 表現對inducible nitric oxide synthase (iNOS) 表現及NO釋放的調控。當以LTA處理不同時間，發現LTA以時間相關的方式刺激COX-2表現及prostaglandin E2 (PGE2) 釋放。LTA所引發之iNOS表現及NO生成可被COX-2抑制劑indomethacin 及NS-398所抑制。且adenylyl cyclase抑制劑dideoxyadenosine及protein kinase A抑制劑KT-5720皆可抑制LTA所引發之iNOS表現及NO釋放。當以PGE2 或dibutyryl-cAMP (cAMP類似物) 處理RAW 264.7 細胞，發現PGE2或dibutyryl-cAMP可以刺激iNOS表現。由electrophoretic mobility shift assay (EMSA) 的結果發現LTA可以時間相關的方式增強NF-kB與DNA結合的能力，當加入indomethacin、NS-398、KT-5720等抑制劑前處理細胞，皆有抑制作用。這表示LTA刺激NF-kB活性的增加可能受到COX-2及protein kinase A的調控。另外LTA也以時間相關的方式活化p44/42及p38 mitogen-activated protein kinase (MAPK)。Indomethacin及NS-398可抑制LTA所引發p44/42 或p38 MAPK之活化，而PGE2及dibutyryl-cAMP可以時間相關的方式刺激p44/42及p38 MAPK活化；且dideoxyadenosine及KT-5720可抑制PGE2所引發p38 MAPK之活化，但p44/42 MAPK則否。由此證明LTA經由刺激COX-2表現、PGE2生成可引發p44/42及p38 MAPK活化，其中p38 MAPK的活化經由PKA-cAMP的活化而來，但p44/42 MAPK則否。綜合以上的結果得知，在RAW 264.7 巨噬細胞中，LTA可刺激COX-2表現而導致PGE2釋放，PGE2釋放後再進一步刺激iNOS表現及NO合成，此PGE2可能以回饋作用的方式引發adenylyl cyclase-cAMP-protein kinase A路徑及NF-kB活化而調控iNOS表現及NO釋放。另外在LTA刺激下，PGE2的釋放也會刺激p44/42及p38 MAPK之活化，而此p38 MAPK之活化是經由adneylylyl cyclase-cAMP-protein kinase A 路徑來調控，但p44/42則否。

  2. Role of Cyclooxygenase-2 Expression on Lipoteichoic Acid-Mediated Nitric Oxide Formation in RAW 264.7 Macrophages • In this study, we investigated the role of cyclooxygenase-2 (COX-2) expression on lipoteichoic acid (LTA)-mediated nitric oxide synthase (iNOS) expression and NO release in murine RAW 264.7 macrophages. LTA caused concentration- and time-dependent increases in COX-2 expression and PGE2 release. LTA also induced iNOS expression and NO release; these events were inhibited by COX-2 inhibitors (indomethacin and NS-398), adenylyl cyclase (AC) inhibitor (DDA) or protein kinase A (PKA) inhibitor (KT-5720). Furthermore, PGE2 and cAMP analog (dibutyryl-cAMP) induced iNOS expression in dose- and time-dependent manners. Moreover, LTA caused time-dependent increases in NF-kB activation; this stimulatory effect was inhibited by indomethacin, NS-398 or KT-5720. These results suggest that LTA induced COX-2 expression and PGE2 release which in turn result in iNOS expression and NO release via the pathway of adenylyl cyclase, PKA and NF-kB. In addition to NF-kB activation, we further investigated the role of PGE2/PKA on the p44/42 and p38 mitogen-activated protein kinase (MAPK) activation caused by LTA. LTA induced p44/42 and p38 MAPK activation. Indomethacin and NS-398 reduced LTA-induced p44/42 MAPK and p38 MAPK activation. PGE2 and dibutyryl-cAMP also resulted in activations of p44/42 and p38 MAPK. However, dideoxyadenosine and KT-5720 reduced the PGE2-induced p38 MAPK activation, but not p44/42 MAPK. We demonstrate that LTA-induced COX-2-dependent PGE2 release and PKA activation contribute to p38 MAPK activation, but not p44/42 MAPK. Taken together, Lta might induce COX-2 expression and PGE2 release which in turn upregulates NF-kB activation, and finally induced iNOS expression and NO release. PGE2 might induce NF-kB activation or p38 MAPK activation via cAMP-PKA, which also resulted in p44/42 MAPK activation without cAMP-PKA pathway.

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