Inhibitory effect of three isoflavonoids from Radix Puerariae on TNF-alpha-induced adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) Abstract: Atherosclerosis (AS) is a chronic and complex vascular inflammatory disease characterized by lipid deposition and even lipid plaque formation in the vascular intima. Both the etiology and the pathology of the disease remain unclear and several mechanisms had been proposed such as the lipid infiltrative theory, the thrombosis hypothesis, the platelet aggregation theory, and the clone hypothesis as the cause of this disease. Now, in general, most researchers believed that the pathogenesis of AS is the complicated results of the interaction among the vascular cells, extracellular matrix, blood constituents (especially monocytes, platelets and low density lipoprotein (LDL)), local hemodynamic environment and heredity. Therefore, there is no single etiological factor for AS. At present, the general view of the pathogenesis of AS is that AS is the complicated interaction among injury, inflammation and immune dysfunction. Due to the complicated pathogenesis of AS which includes many links and drug targets, the development of effective drugs which act on these multiple specific targets for AS prevention and therapy is slow unavailable. Preclinical and clinical studies revealed that traditional Chinese medicine were comprised of multiple chemical components and have mutli- therapeutic targets and showed good therapeutic effect in AS prevention and therapy. However, the action mechanisms of most Chinese medicine were still unclear yet. More reports suggested that Gegen (Radix Puerariae)., a Chinese medicine showed good anti-atherosclerosis effects but the underlying mechanism of action need to be elucidated. In this manuscript, HUVECs were used to explore the possible anti-atherosclerosis mechanisms of action of isoflavonoids including daidzein, daidzin and puerarin from Gegen. The effects of daidzein, daidzin and puerarin on TNF-a induced ICAM-1 and VCAM-1 expression were tested by ELISA, immunofluorescence staining and Western blotting. Their inhibitory effects on cell-cell adhesion between endothelial cells and monocytes were also determined. Results showed that no significant cytotoxicity was observed for treatment with 1-100µM puerarin. At 1-30µM neither daidzein nor daidzin showed cytotoxicity to endothelial cells while both of them showed obvious cytotoxicity at 100µM. Cell-ELISA results revealed that all of these three isoflavonoids compounds from Gegen could significantly inhibited TNF-a induced ICAM-1 expression in, a dose-dependent manner. Immunofluorescence staining and Western blotting showed similar results as observed in cell-ELISA. In summary, all these results suggested that Gegen (Radix Puerariae) may reduce atherosclerosis through inhibiting endothelial ICAM-1 and VCAM-1 expression. Keywords: Puerarin, Daidzin, Daidzein, HUVEC, Anti-atherosclerosis.

摘要 動脈粥樣硬化(Atherosclerosis, AS)是以富含脂肪的斑塊在大動脈壁聚積 為特徵的系統病變,是心腦血管病的主要病理基礎。動脈粥樣硬化形成原因相當 複雜,現代醫學關於AS的病因較一致的看法是由損傷、炎症、免疫功能障礙三者 相結合作用的結果。其主要臨床表現是心肌梗塞、中風和外周血管疾病,常伴 有高血壓、高膽固醇血症或糖尿病等。動脈粥樣硬化導致的疾病是發達國家第一 位的死亡原因[12]。在我國,隨著經濟社會的發展和人口的老齡化,心腦血管病發 病率與死亡率近年也顯著增加。 中藥含有的諸多成分,具有多環節、多靶點作用的性質,對機制複雜的動脈 粥樣硬化有更好更全面的防治作用。越來越多的報導說明中藥葛根具有抗動脈粥 樣硬化的藥理作用,但在葛根怎樣抗動脈粥樣硬化作用上還沒有清晰的闡釋。本 第5頁共63頁 5 澳門大學碩士學位論文 文以人臍靜脈內皮細胞(HUVEC)作體外模型詳細描述葛根異黃酮類提取物是如何 抗動脈粥樣硬化。首先以MTT細胞計數的方法篩選出葛根異黃酮類化合物作用於 HUVEC的有效濃度,並同時完成毒性篩選。在細胞黏附實驗中,葛根提取物對THP-1 細胞與HUVEC的黏附作用有明顯抑制。在ELISA實驗中,檢測出大豆苷元 (daidzein)、大豆 (daidzin)、葛根素(puerarin) 三種主要異黃酮類提取 物具有明顯的抑制炎症因數ICAM-1等的表達的作用。在western blot實驗中,檢 測出大豆苷元(daidzein)、大豆苷(daidzin)、葛根素(puerarin)低、中、 高濃度對炎症因數ICAM-1等的表達有不同程度的抑制。另外,還通過螢光顯微觀 察,進一步證實葛根提取物對ICAM-1等炎症因數表達的抑制作用。以上研究表明 葛根提取物具有抗動脈粥樣硬化的作用。 關鍵字:葛根,異黃酮類提取物,人臍靜脈內皮細胞,抗動脈粥樣硬化