數種植物性雌激素對動脈粥狀硬化相關因子之影響

The Effects of Several Phytoestrogens on Atherosclerosis related Factors

沈靈(Ling Shen)

指導教授 ： 吳文惠 ； 蔡帛蓉

國立臺灣師範大學/教育學院/人類發展與家庭學系/碩士(2007年)

摘要

動脈粥狀硬化是很複雜的血管病變，好發於冠狀動脈。從早期血管腔的內皮層下方出現脂肪條堆積，到出現動脈硬化斑塊等複雜病灶過程需數十年，導致心血管疾病的發生。
許多臨床研究指出大豆異黃酮對心血管具有保護作用，且可能與雌激素活性有關。本研究探討多種含植物性雌激素活性食材之萃物 (苜蓿芽、山藥乙酸乙酯萃物，茉莉、菊花甲醇萃物) 或純化合物 (enterolactone，sesamin，genistein) 是否能抑制： (1) 已分化的THP-1細胞在氧化型低密度脂蛋白刺激下，促發炎介質（IL-1β、TNF-α、MCP-1）與基質金屬蛋白?MMP-9的mRNA表現及蛋白質分泌量 (2) 已分化的THP-1細胞在氧化型低密度脂蛋白刺激下，攝入膽固醇的程度以及其CD36與ABC A1 mRNA表現量。(3) 內皮細胞在氧化型低密度脂蛋白刺激後，與THP-1 細胞黏附的程度。
實驗設計：(1) 將THP-1細胞以PMA誘導細胞完全分化後，加入食材萃物或純化合物，先培養兩小時後，再給予氧化型低密度脂蛋白處理細胞共同培養24或48小時，收集上清液以ELISA分析IL-1β、TNF-α、MCP-1與MMP-9的含量，並且經過共同培養16或48小時，收集細胞以分析IL-1β、TNF-α及MMP-9之mRNA表現。(2) 將已分化THP-1細胞加入食材萃物或純化合物，先培養兩小時後，再給予氧化型低密度脂蛋白處理細胞共同培養16或48小時，收集細胞以分析其總膽固醇含量及CD36與ABCA1之mRNA表現。(3) 將內皮細胞 (HAECs) 加入各種食材萃物或化學物與氧化型低密度脂蛋白共同處理24小時，再加入已染有螢光的THP-1 細胞共同培養後，分析細胞黏附的程度。
結果發現：(1)所有測試物皆顯著下降IL-1β的濃度；除了enterolactone外，所有測試物皆顯著下降TNF-α濃度；enterolactone，sesamin，genistein及苜蓿芽乙酸乙酯萃物顯著下降MCP-1濃度；enterolactone，genistein，苜蓿芽乙酸乙酯萃物及山藥乙酸乙酯萃物能顯著下降MMP-9濃度與活性。(2) enterolactone、sesamin、茉莉甲醇萃物和菊花甲醇萃物能顯著下降細胞內總膽固醇濃度，且茉莉和菊花甲醇萃物能顯著下降CD36 mRNA表現。 (3)所有測試物皆可顯著降低內皮細胞與單核球THP-1黏附的程度，其中sesamin、苜蓿芽乙酸乙酯萃物及菊花甲醇萃物還能顯著下降MCP-1濃度。
根據以上結果推測，茉莉與菊花甲醇萃物可能經由抑制CD36表現，降低巨噬細胞內總膽固醇量，而具有抑制泡沫細胞生成之潛力。所有測試物除了enterolactone外，皆可顯著下降IL-1β及TNF-α濃度，可能具抗發炎之效用。Enterolactone、genistein及苜蓿芽乙酸乙酯萃物皆能顯著抑制MCP-1與MMP-9濃度及MMP-9活性，推測他們可能具有減少單核球聚集、下降單核球與內皮細胞黏附的程度、減少平滑肌細胞移行，及減緩斑塊破裂的可能潛力。
總結以上發現，enterolactone、genistein、山藥乙酸乙酯萃物與苜蓿芽乙酸乙酯萃物皆可能經由抑制初期的細胞黏附，抑制促發炎介質分泌，並增加後期的斑塊穩定性，具有減緩動脈粥狀硬化之潛力。

關鍵字

動脈粥狀硬化 ； 巨噬細胞 ； 促發炎介質 ； 植物性雌激素 ； 內皮細胞

並列摘要

Atherosclerosis is now considered to be an inflammatory disease of the blood vessel wall, characterized in early stages by endothelial dysfunction, recruitment and activation of monocyte/macrophages, and dedifferentiation and migration of vascular smooth muscle cells (VSMCs) to later form the bulk of the atherosclerotic plaque. Many clinical studies indicate that dietary soy isoflavones exhibit the atheroprotective effects through their estrogen activity. The aim of this study was to investigate the effects of botanical extracts (ethyl ester extracts of yam and alfalfa & methanolic extracts of jasmine and chrysanthemum) and chemicals (enterolactone, sesamin and genistein), which possess potential estrogen activity, on (1) cellular cholesterol accumulation and gene expression of CD36 and ABCA1, (2) the concentration and gene expression of proinflammatory mediators (IL-1β, TNF-α, and MCP-1) and MMP-9 in oxidized LDL (oxLDL)-stimulated THP-1 macrophages; (3) the adhesion of monocytic THP-1 cells to oxLDL-treated human aorta endothelial cells (HAECs).
THP-1 derived macrophages activated by PMA were treated with oxLDL for 2 days to induce lipid uptake by macrophages and establish a foam cell-like phenotype. The cells were co-incubated with the botanical extracts and chemicals for 24~48 hr. The concentrations of IL-1β, TNF-α, MCP-1 and MMP-9 in cultural supernatants were determined by ELISA method. To evaluate the monocyte adhesion to endothelial cells, HAECs were pretreated with the test compounds and oxLDL for 24 hours and then co-cultured with fluorescence-labeled monocytic THP-1 cells. The monocyte adhesion was determined by measuring the fluorescent intensity.
These results showed that enterolactone, sesamin, jasmine and chrysanthemum extracts significantly decreased the cellular cholesterol content. Enterolactone, genistein, jasmine and chrysanthemum extracts significantly inhibited the expression of CD36 after THP-1 macrophages incubated with oxLDL for 16hrs. All of the chemicals significantly inhibited the expression of CD36 after THP-1 macrophages incubated with oxLDL for 48hrs.
All of the test compounds significantly inhibited the secretion of IL-1β. Sesamin, genistein and the extracts of yam, alfalfa, jasmine and chrysanthemum significantly decreased the concentration of TNF-α. All of the chemicals and alfalfa extract inhibited the secretion of MCP-1. Yam and alfalfa extracts, enterolactone and genistein decreased the production and the activity of MMP-9. All of the test compounds significantly inhibited the adhesion of THP-1 monocytes to HAECs.
These results suggest that enterolactone, sesamin, jasmine and chrysanthemum extracts may reduce cellular cholesterol via the inhibition of CD36 expression. All of the chemicals and extracts may prevent atherogenesis, probably via its anti-inflammatory function by reducing the production of pro-inflammatory cytokines and the adhesiveness between monocytes and endothelial cells.

並列關鍵字

atherosclerosis ； macrophage ； proinflammatory mediators ； phytoestrogens ； endothelial cell

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