在各種發炎疾病狀態下一氧化氮 (nitric oxide ； NO) 是一個重要調節及作用的分子。組織在發炎時，大量的 NO 會藉由誘導型一氧化氮合成酵素 (inducible nitric oxide synthase ； iNOS) 產生。尤其在內毒素所引起敗血性休克時， iNOS 產生過量的 NO 被認為是導致低血壓，低反應性血管收縮及組織氧化性傷害的主要原因之一。本篇論文主要探討，抗氧化強度為 a-tocopherol 7 倍之 octyl caffeate 對於脂多醣體 (LPS) 及干擾素 -g (IFN-g) 在大鼠動脈平滑肌細胞產生 iNOS 的影響，且進一步探討這其中所牽涉的抑制機制。在老鼠動脈平滑肌細胞中，我們發現單以 IFN-g (100 U/ml) 處理細胞，無法使平滑肌細胞釋放 NO ，然而 LPS (100 mg/ml) 能明顯誘導 NO 的產生。若同時將平滑肌細胞投予 LPS 及 IFN-g 則會隨著濃度和時間的增加而明顯提高 NO 之釋出。當平滑肌細胞於 LPS (100 mg/ml) 和 IFN-g (100 U/ml) 合併處理 24 小時後，在培養液中可測得之 NO 含量約增加為未加藥組的 8 倍。以不同濃度的 octyl caffeate 處理後，平滑肌細胞會明顯有意義且以劑量方式減少 LPS 及 IFN-g 誘發產生的 NO 。除此之外，事先投予 octyl caffeate (1 、 10 and 50 mM) 30 分鐘後，再以 LPS 及 IFN-g 處理 24 小時，也會隨劑量增加而抑制 iNOS mRNA 及 iNOS protein 的表現量。同時，以高濃度 50 mM 之 octyl caffeate 處理平滑肌細胞 24 小時，並不影響細胞的存活。實驗更進一步發現 octyl caffeate 並不影響 IkB-a 的降解作用，但卻會抑制同屬 MAPK 成員的 JNK/SAPK 及 ERK1/2 這兩個蛋白質的磷酸化。綜合以上的發現，我們推測 octyl caffeate 為一強抗氧化劑，其抑制 LPS 及 IFN-g 誘發初代大鼠動脈平滑肌細胞釋放 NO 的作用機轉可能是透過減少 JNK 及 ERK1/2 的活化，接著抑制 iNOS mRNA表現，最後導致 iNOS protein 減少，進而減少 NO 的累積。這個結果提供了 octyl caffeate 抑制平滑肌細胞 iNOS 之作用機轉。因此未來可能利用 octyl caffeate 抑制 iNOS 的作用來治療敗血症引發的心血管系統發炎性傷害。

天然咖啡酸衍生物 octyl caffeate抑制內毒素 /干擾素引發平滑肌細胞產生誘導型一氧化氮合成酵素之機轉探討

Nitric oxide (NO) is an important regulator and effector molecule in various inflammatory disease states. High output of NO during inflammation is generated by the inducible isoform of nitric oxide synthase (iNOS). Overproduction of NO by iNOS is thought to be the principal factor of hypotension and hyporeactivity to vasoconstrictor agents observed in endotoxic shock.The present study was to investigate the effect of potent antioxidant, octyl caffeate, on the induction of iNOS formation by lipopolysaccharide (LPS) and interferon-g (IFN-g) in cultured rat aortic smooth muscle cells (RASMC). According to our study, in primary RASMC cultures, IFN-g (100 U/ml) alone did not stimulate NO production, while LPS (100 mg/ml) did. And IFN-g combined with LPS could dose-dependently (LPS: 30, 50, 100 mM) and time-dependently (4 - 24 hr) induce NO production. LPS (100 mg/ml) and IFN-g (100 U/ml) synergistically induced a 8-fold production of NO on RASMC for 24 hrs incubation. Octyl caffeate caused a significant and concentration-dependent inhibition on the production of NO upon stimulation by LPS (100 mg/ml) and IFN-g (100 U/ml) in RASMC.In addition, RASMC pretreated with octyl caffeate (1, 10, 50 mM) by stimulation of LPS/IFN-g caused a concentration-dependent reduction both in iNOS mRNA and protein expression. At the same dose, octyl caffeate did not significantly reduced the cell viability. Furthermore, we found that octyl caffeate did not inhibit IkB-a degradation. In addition, octyl caffeate (10 and 50 mM) significantly inhibited JNK/SAPK and ERK1/2 phosphorylated activation.Therefore, based on the above observations, we suggested that antioxidant, octyl caffeate diminished LPS/IFN-g-induced NO production in RASMC by a mechanism involving inhibition of MAPK activation, followed by the inhibition of iNOS mRNA, thereby leading to the inhibition of iNOS protein and NO production. These results suggest a possible role of octyl caffeate in managing septic inflammation of cardiovascular system through inhibition of iNOS induction.

Mechanisms of octyl caffeate involved in the inhibition of LPS/interferon-g-induced iNO

synthase expression in rat aortic smooth muscle cells