紅鳳菜乙醚萃取物對於人類血管內皮細胞間移行作用之影響

Effects of ether extract of Gynura bicolor on endothelial transmigration in human endothelial cells

蘇冠華(Kuan-Hua Su)

指導教授 ： 吳志忠

長榮大學/健康科學學院/醫學研究所/碩士(2013年)

https://doi.org/10.6833/CJCU.2013.00135

摘要

心血管疾病發病的主要原因之一即為動脈粥狀硬化。發病早期病程中，白血球細胞將分化成巨噬細胞，侵入到內皮細胞下層引起一連串的動脈粥狀硬化病理變化，此過程稱之為白血球細胞的移行作用 (transmigration)。移行作用的過程可分為三個步驟，包括：細胞滾動 (rolling)、黏附 (adhesion) 及侵入 (invasion) 作用。如能利用日常飲食之食材，在平日攝食中達到抑制白血球細胞的移行作用，將有助於預防動脈粥狀硬化之發生。紅鳳菜為民間常食用之蔬菜，民眾多認為紅鳳菜具有活血、消腫、解毒及促進傷口癒合等之功效。在過去研究中已知紅鳳菜乙醚萃取物 (Gynura bicolor ether extracts, GBEE) 具有抗發炎以及抑制黏附蛋白 ICAM (intercellular adhesion molecule, ICAM) 之生成，因此，本研究擬進一步探討 GBEE 對於細胞移行作用之影響，以進一步瞭解 GBEE 預防動脈粥狀硬化之作用。本研究分為二大部分，首先分別利用細胞之黏附及侵入作用之指標，探討 GBEE 調控內皮細胞黏附及侵入之最佳濃度。第二部分則探討最適濃度之 GBEE 及其相對含量之主要成分對於細胞黏附及侵入作用之影響及其調控路徑。期望藉由本研究之完成能瞭解 GBEE 及其活性成分如何調節細胞之移行作用及其調控機轉。本研究之第一部分為預先以 10、50 或 100 μg/mL GBEE 處理人類內皮細胞株 (human umbilical vein cell line; EA. hy926 cells) 8 小時後，再合併 10 ng/mL TNF-α 共同培養 3 小時，經由分析細胞生存能力、細胞黏附能力、細胞間通透性、細胞之侵入作用相關連結調控蛋白之變化，以瞭解 GBEE 是否具有降低動脈粥狀硬化之作用。由實驗結果顯示， 100 μg/mL GBEE 可顯著抑制細胞黏附作用、細胞間通透性、以及連結蛋白磷酸化內皮細胞鈣黏蛋白 (phosphorylation vascular endothelial, p-VE-cadherin)、血小板/內皮細胞附著蛋白 (platelet endothelial cell adhesion molecule, PECAM) (p<0.05) 而降低內皮細胞黏附及侵入作用。經 GBEE 主要活性成分分析後，將 100 μg GBEE 換算其主要成分及含量分別是 β-胡蘿蔔素為 7.46 mg/g、葉綠素為 11.5 mg/g 及槲皮素為 25.3 μg/g。接下來以此三種成分、兩者合併 (β-胡蘿蔔素合併葉綠素、β-胡蘿蔔素合併槲皮素、葉綠素合併槲皮素)，或是三者合併 (β-胡蘿蔔素、葉綠素合併槲皮素) 為處理組，分別探討此等成份對黏附及侵入作用之影響。由實驗結果顯示 GBEE、葉綠素、β-胡蘿蔔素合併葉綠素、β-胡蘿蔔素合併槲皮素、葉綠素合併槲皮素以及 β-胡蘿蔔素、葉綠素合併槲皮素處理組皆可抑制細胞黏附作用 (p<0.05)。細胞侵入實驗分析中，除了槲皮素組外，皆可抑制細胞侵入作用；在相關連結蛋白質之表現實驗中 β-胡蘿蔔素、葉綠素及 β-胡蘿蔔素合併槲皮素皆可抑制磷酸化 VE-cadherin 之生成 (p<0.05)。β-胡蘿蔔素、葉綠素合併槲皮素處理內皮細胞可降低旁細胞路徑上連結蛋白 PECAM 之表現 (P<0.05)。 β-胡蘿蔔素、葉綠素、槲皮素、葉綠素合併槲皮素及 β-胡蘿蔔素、葉綠素合併槲皮素可降低磷酸化 Src 蛋白質之生成 (p<0.05)。100 μg/mL GBEE 可降低 ROS 之生成 (p<0.05)。β-胡蘿蔔素合併槲皮素、葉綠素合併槲皮素及 β-胡蘿蔔素、葉綠素合併槲皮素皆可降低移行作用之跨細胞路徑上的 ROCK1 蛋白質之生成 (p<0.05)。由以上結果顯示 GBEE 可抑制旁細胞路徑上 p-VE-cadherin、PECAM 之生成及降低在跨細胞路徑上 Rho A 蛋白質之生成，達到抑制細胞黏附及侵入之作用。總結， 100 μg/mL GBEE 可經由降低內皮細胞之黏附及侵入作用而具有抑制細胞移行作用之效用，但是效果不及 β-胡蘿蔔素、葉綠素合併槲皮素之組別。GBEE 主要活性成分中抑制內皮細胞之移行作用可能是來自 β-胡蘿蔔素合併葉綠素、葉綠素合併槲皮素以及 β-胡蘿蔔素、葉綠素合併槲皮素等組合。

關鍵字

內皮細胞鈣黏蛋白 ； 血小板/內皮黏附蛋白 ； 動脈粥狀硬化

並列摘要

Atherosclerosis is one of the major cause of cardiovascular disease (CVD). Cell transmigration, progressive activation of atherosclerosis, are including WBC rolling, adhesion and invasion on endothelial cells. Using nature dietary materials to suppression transmigration is nice for atherosclerosis prevention. Gynura bicolor is a common vegetable in Taiwan and Asia. The populace consider Gynura bicolor has activating blood, wound healing. The aim of in this study is want to know effect of Gynura bicolor on atherosclerosis prevention. Human endothelial cells (EA. hy926 cells) cultured as a experimental model and Gynura bicolor ether extracts (GBEE) and its major components as experimental materials in this study. First, to study effects of various concentration of GBEE on adhesion and invasion, two major process of transmigration. EA. hy926 cells were pretreatment with 10, 50 or 100 μg/mL GBEE for 8 hours, then co-treated with 10 ng/mL TNF-α for 3 hours to observe cell viability、cell permeability、cell adhesion and junction protein expression. Result shows that 100 μg/mL GBEE can inhibit cell adhesion, cell permeability, and p-VE-cadherin、PECAM protein expression (p<0.05). According to the result of GBEE composition analysis, 100 μg/mL GBEE including : 7460 μg/g β-carotene、11500 μg/g chlorophyll and 25.3 μg/g quercetin, respectively. Therefore, 100 μg/mL GBEE, β-carotene, chlorophyll, quercetin, β-carotene combined chlorophyll, β-carotene combined quercetin, chlorophyll combined quercetin and β-carotene combined chlorophyll and quercetin, respectively, were as experimental materials in next stage of this study. EA. hy926 cells were pretreatment with above experimentals for 8 hours, then co-treated with 10 ng/mL TNF-α for 3 hours to observe cell viability、cell permeability、cell adhesion and junction protein expression. Result show that β-carotene combined chlorophyll, β-carotene combined chlorophyll, β-carotene combined quercetin, chlorophyll combined quercetin and β-carotene combined chlorophyll and quercetin , respectively, all can inhibit cell adhesion and cell invasion on EA. hy926 cells (p<0.05). Quercetin and β-carotene combined chlorophyll can reduce PECAM expression of EA hy926 cells (p<0.05). β-carotene, chlorophyll, chlorophyll combined quercetin and β-carotene combined chlorophyll and quercetin, respectively, all can reduce phosphorylation Src protein expression of EA hy926 cells (p<0.05). 100 μg / mL GBEE can reduced ROS levels of EA. hy926 cells (p<0.05). β-carotene combined quercetin、chlorophyll combined quercetin and β-carotene combined chlorophyll and quercetin, respectively, all can reduce ROCK1 protein expression of EA. hy926 cells (p<0.05). These results indicated that GBEE and its major ingredients were suppression paracellular invasion through inhibit inhibit ICAM, p-VE-cadherin, PECAM protein expression, and suppression transcellular invasion through reduce Rho A protein expression. Gynura bicolor can prevent atherosclerosis through the inhibit cell transmigration.

並列關鍵字

Gynura bicolor ； p-VE-cadherin ； PECAM ； atherosclerosis

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