摘要或動機

動脈硬化是個致病率和致死率相當高的慢性發炎疾病。HMG-CoA 還原酵素抑制劑 纇藥物，簡稱statin, 是一類強效的降血脂藥物，而且在臨床上對於心血管疾病有廣泛的 治療效果。然而近年來的研究報導指出，statin 會有如此廣效治療效果，其原因不僅僅 是因為它的降血脂能力，而是statin 也具有抑制發炎作用的效果。雖然臨床上已經證實 statin 可以減緩動脈硬化的病程，但是statin 對於誘發型一氧化氮合成酵素(iNOS)表現的 調控機制還不明確。有文獻指出NOS 代謝產物nitric oxide (NO)可以改善血流，而可能 在動脈硬化上扮演保護角色。值得一提的是雖然適量 NO 有維持血管恆定的功能，過 量時則會造成血壓過低休克的現象，這就是細菌感染後因內毒素 lipopolysaccharide (LPS) 作用引發敗血性休克的主要原因之一。在本實驗中，我們使用fluvastatin、lovastatin、 pravastatin 和 atorvastatin 這四種statins 來探討它們對於血管平滑肌細胞由LPS 及IL-1β 誘導iNOS 基因表現的影響。我們發現，statin 可以抑制LPS 所誘發的NO 和iNOS 表現， 但卻會促進IL-1β所誘發的反應。NF-κB 在iNOS 的基因調控上扮演重要的角色，而在 探討NF-κB 被LPS 和IL-1β活化的情形中，statin 同樣會促進IL-1β活化NF-κB，但抑制 LPS 活化NF-κB。我們也發現fluvastatin 對於IL-1β所引發NO 的產生、iNOS 的表現、 NF-κB 的活化，以及p65 向細胞核移動的促進作用，在ROCK 抑制劑Y-27632 的處理後 可以看到相同的現象。IKK kinase assay 顯示Y-27632 對於LPS 所促進的IKK 活性影響 很小，但是會促進IL-1β的活化作用。接著，在ROCK 的活性方面，LPS 會抑制原本已 表現的ROCK 活性， 而相反的，IL-1β會增加ROCK 的活性。總括來說，這些結果顯示 ROCK 在血管平滑肌細胞中扮演IKK/NF-κB 的負向訊息調控者角色，而這個機制在LPS 和IL-1β的訊息傳遞路徑中有不同的調控。即ROCK 以負向調控機制角色影響IL-1β的 訊息傳遞，卻不存在於LPS 的情況中。就是因為透過反轉這個負向調控的機制，statins 3 和ROCK 抑制劑對於LPS 及IL-1β引發血管平滑肌細胞的iNOS 表現，呈現相反的調控 結果。這些作用可能參與statin 預防血管再阻塞，抗發炎，抗動脈硬化的作用。此外Statin 抑制LPS 的iNOS 表現作用或許將來可運用於治療敗血性休克。利用基因微陣列分析也 偵測到一些受fluvastatin 正向或負向調控的基因，目前我們正朝鑑定基因的表現改變及 確認其功能，生理意義進行實驗中。 The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, statins, are potent inhibitors of cholesterol synthesis and have wide therapeutic use in cardiovascular diseases. Recent evidence, however, suggests that the beneficial effects of statins may extend beyond their action on serum cholesterol levels. Although statins have been shown to reduce progression of atherosclerosis, little is known about mechanism by which statins affect iNOS expression. Optimal level of NOS product, NO, possesses the anti-inflammation and anti-proliferation effects in atherosclerosis, while large amount of NO contributes to the septic shock in response to bacterial endotoxin, lipopolysaccharide (LPS). In this study, we investigated the effects of fluvastatin, lovastatin, pravastatin and atorvastatin on IL-1β- and LPS-induced NO production in cultured rat vascular smooth muscle cells (VSMC). We found statins can inhibit LPS-induced iNOS expression and NO production, while they can potentiate IL-1β-elicited responses. In studying the activity of NF-κB, which plays an important role for iNOS gene induction, we found that fluvastatin can increase IL-1β-induced p65 nuclear translocation and NF-κB activity, while inhibit those induced by LPS. The potentiation effects of fluvastatin on IL-1β-induced NO production, iNOS expression, NF-κB activation and p65 nuclear translocation were all mimicked by a ROCK inhibitor, Y-27632. IKK kinase assay showed that Y-27632 itself has minimal effect on LPS-induced IKK activation, while enhances the response of IL-1β. Studies on examining ROCK activity showed LPS can downregulate constitutive ROCK activity, while IL-1β oppositely increases ROCK activity. Taken together these data suggest ROCK is a crucial negative regulator of IKK/NF-κB signaling pathway in VSMC, and this negative control is existing in the action IL-1β, but is absent in the action of LPS. Through abrogating the function of this negative regulator, statins and ROCK inhibitor thus differentially regulate iNOS expression induced by LPS and IL-1β in VSMC. These results suggest that stimulation of iNOS expression in the presence of IL-1β might contribute to the beneficial effects of statins in atherosclerotic process in terms of vasodilation, anti-inflammation and inhibition of smooth muscle cell proliferation. In contrast, the diminishing effect on LPS-induced NO response possibly may provide new therapeutic strategy in sepsis. Al these results strengthen the pleiotropic actions of statins in anti-inflammation and anti-atherosclerosis. Preliminary microarray analysis further revealed several genes either upregulated or downregulated by fluvastatin. The identification of these genes and studying their functional roles in atherosclerosis are currently in progress.