劉凱莉洪奇楠Chi-Nan Hung營養學研究所2026-03-249999/12/312014https://handle.ncl.edu.tw/11296/a9va59https://scholars.csmu.edu.tw/handle/123456789/6948依據全球心血管疾病流行病的調查顯示，全世界5000 萬的死亡人口中，有4000 萬的人口是發生在發展中國家。根據2012 年台灣行政院衛生署國人十大死因調查結果，惡性腫瘤28.4%是首位，心臟疾病（高血壓性疾病除外，11.1%）和腦血管疾病(7.2%)分別排列第二和第三死因。動脈血管硬化是一種長期發炎性疾病，也是心血管疾病的主要成因。動脈血管硬化最驚人的結果是心臟病發作和中風。動脈血管硬化肇因於氧化修飾的脂蛋白、單核球衍生的巨噬細胞、T-淋巴球以及動脈血管壁上的正常細胞之間的交互作用。另一方面，動脈血管硬化牽涉許多不同的細胞，其中包括巨噬細胞、淋巴球、嗜中性球、內皮細胞以及平滑肌細胞。防止促進動脈血管硬化的發生，是目前一項非常重要的健康議題。基於以上原因，本研究主要目地在利用具有保護效應的蘿蔔硫素(sulforaphane , SFN)來探討它對TNF-α 誘發內皮細胞發炎反應的影響，接著進一步探討利用TNF-α 誘發平滑肌細胞增生、遷移和訊息傳遞的效應與機轉。蘿蔔硫素是一種異硫氰酸酯(isothiocyanates)，富含在十字花科蔬菜中，例如花椰菜、甘藍菜、青花椰菜和青花椰菜芽等。過去研究指出，蘿蔔硫素具有抗癌、抗發炎、抗氧化等多樣的生理效用。首先，應用TNF-α 刺激觸發內皮細胞中發炎調解因子的分泌，以塑造動脈血管硬化的模式。利用蘿蔔硫素預處理，再以TNF-α 刺激ECV 304 內皮細胞，是否影響內皮黏附分子(intercellular adhesion molecule-1 ; ICAM-1)的表現。結果顯示，以TNF-α 刺激ECV 304 內皮細胞，利用蘿蔔硫素可減弱內皮黏附分子的表現，蘿蔔硫素抑制前發炎細胞激素如IL-1β, IL-6 和 IL-8 的作用是呈現劑量關係，同時是透過抑制NF-κB DNA 結合活性。另外，蘿蔔硫素減少了TNF-α 刺激所產生的IκB kinase (IKK) 和 IκBα, Rho A, ROCK, ERK1/2 以及PAI-1 磷酸化作用。綜合以上，蘿蔔硫素抑制了TNF-α 處理下內皮黏附分子表現，和NF-κB DNA 結合活性以及在內皮細胞中，是透過抑制Rho A/ROCK/ NF-κB 訊息途徑，所以結果顯示，動脈血管硬化損傷中，蘿蔔硫素有抑制前發炎作用。在平滑肌細胞研究方面，我們探討利用蘿蔔硫素對於TNF-α 處理的A7r5 平滑肌細胞增生、移動和訊息傳遞作用的影響，在傷口癒合實驗(Wound healing assay) 和細胞趨性分析實驗(Boyden chamber assay)中，蘿蔔硫素抑制TNF-α 處理的A7r5 平滑肌細胞的移動作用，而且，蘿蔔硫素降低基質金屬蛋白酵素-9 (Matrix Metalloproteinases，MMP-9)表現，並呈現劑量關係。在螢光phalloidin 染色實驗，蘿蔔硫素抑制F-actin 表現，同時，蘿蔔硫素減弱Ras 和RhoA/ROCK 相關蛋白表現，以上結果顯示，蘿蔔硫素可減弱MMP-9 調控A7r5 平滑肌細胞移動作用和抑制Ras 和RhoA 的訊息傳遞路徑。According to the global epidemiologic survey, a total of the 50 million died in atherosclerotic cardiovascular disease. Among those, 40 million occur in developed and developing countries. The Department of Health, Executive Yuan of Taiwan reports that cardiovascular disease(Hypertension Disease exclude, 11.1%) and cerebrovascular disease (7.2%) are the second and the third cause of death in 2012. The etiology of atherosclerosis is multiple factors and the chronic inflammation disease plays a crucial role. The most devastating consequences ofatherosclerosis are heart attack and stroke. Atherosclerosis results from not only the oxidation-modified lipoproteins, but also the interaction among numerous types of cells including vascular endothelial cells, vascular smooth muscle cells, monocyte-derived macrophages, lymphocytes and neutrophils, and also cross-talk among the cellular elements of the arterial wall. To date, preventing the development and advancement of atherosclerotic lesions is an important public healthissue. Based on these reasons, in this study, we employ two cell models,TNF-α-induced vascular endothelial cells and TNF-α-induced vascular smooth muscle cells, to investigate the cardio-protective effects of sulforaphane (SFN) and the possible mechanisms involved. Sulforaphane(1-isothiocyanate-4-methylsulphinylbutane; SFN) is a sulfur-contained phytocompound that is rich in cruciferous vegetables, such as cauliflower, cabbage, broccoli and broccoli sprouts. Evidence indicates that sulforaphane possesses a diverse biological activity including anti-cancer, anti-inflammation and anti-oxidation effects.Part one; we investigated whether SFN affected the expression of intracellular adhesion molecule-1 (ICAM-1) in TNF-α-induced ECV 304 endothelial cell. Our data showed that SFN attenuated TNF-α-induced expression of ICAM-1 in ECV 304. Pretreatment of ECV 304 cells with SFN inhibited dose-dependently the secretion of proinflammatorycytokines, such as IL-1β, IL-6 and IL-8. SFN inhibited TNF-α-induced NF-κB DNA binding activity. Furthermore, SFN decreased TNF-α-mediated phosphorylation of IκB kinase (IKK) and IκBα, Rho A, ROCK, ERK1/2 and PAI-1 levels. Collectively, SFN inhibited the NF-κB DNA binding activity and downregulated the TNF-α-mediated induction of ICAM-1 in endothelial cells by inhibiting the Rho A/ROCK/ NF-κBsignaling pathway. Therefore, suggesting the beneficial effects of SFN on suppression of proinflammation within the atherosclerotic lesion. Part two; we investigate the capacity of SFN on TNF-α-induced proliferation, migration and signal transduction in A7r5 smooth muscle cells. SFN inhibited the migration ability of A7r5 cells induced by TNF-α using wound healing assay and Boyden chamber assay. After treatment of SFN at various concentrations with TNF-α, A7r5 cell reduced matrix metalloproteinase-9 (MMP-9) expression. Fluorescent phalloidin staining demonstrated that increased intense F-actin staining in theTNF-α-induced A7r5 cells was inhibited by SFN. The levels of Ras and RhoA/ROCK-related proteins in the TNF-α treated A7r5 cells were increased, whereas these protein expression were attenuated by SFN. The results suggested that SFN could mediate A7r5 cells migration by reducing MMP-9 activity involving the suppression of the Ras and RhoAsignaling pathways.thesis