Abstract Effect of Saponins from Panax notoginseng on TNF-alpha-induced Adhesion Molecule Expression in Human Coronary Artery Endothelial Cells By Wang Nan Thesis Supervisor: Dr. Simon Ming Yuen LEE Chinese Medicinal Science Atherosclerosis (AS), a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries, is the cause of the majority of cardiovascular disease and causes more global death and disability than any others. AS can cause cardiovascular and cerebrovascular diseases by blocking large and medium-sized arteries, and finally, it can lead to heart, brain, and other organs and tissues ischemic necrosis, myocardial infarction, and brain death. AS is also called as number one killer of endangering human health and lives in China in recent years. Panax notoginseng, as a commonly used "blood circulation promoting" herb in Traditional Chinese Medicine, is a potential candidate for the purpose. The pathogenesis of atherosclerosis involves a complex series of events, many pathologists raised hypothesis and considered AS as a chronic inflammatory process, with the formation of atherosclerotic plaque as the end results. The endothelial dysfunction, migration and proliferation of smooth-muscle cells, formation of necrotic core and fibrous-cap formation may form the advanced, complicated lesion of atherosclerosis. Panax notoginseng, commonly used in the prescriptions of Traditional Chinese Medicine for blood circulation promotion and to be a potential candidate for the disease cure. However, the biological effects of saponins extract from Panаx notoginseng on atherosclerosis, specifically vascular inflammation and the underlying mechanisms are yet to be fully elucidated. This investigation describes the anti-inflammatory effects of three saponin fractions (total saponin fraction "PNS", protopanaxadiol-type saponin fraction "PDS" and protopanaxatriol-type saponin fraction "PTS"), amd two purified ginsenosides (Rg1 and Rb1) on Human Coronary Artery Endothelial Cells (HCAECs) in vitro. As we known, elevated levels of a inflammatory cytokine, such as tumor necrosis factor-α (TNF-c), can adversely affect the arterial wall and induce the inflammatory response. Thus, TNF-c-stimulated HCAECs was used as a vascular inflammatory model in this study. The three saponin fractions (PNS, PDS and PTS) and two purified ginsenosides (Rbl and Rg1) were identified to decrease the THP-1 monocyte cells adhesion to HCACEs stimulated with TNF-a by a cell-cell adhesion assay. In order to identify mechanism underlysing the suppressive effect of constituents from Panax notoginseng on TNF-a stimulated cell-cell adhesion, the change of cell surface expression of adhesion molecules, ICAM-1 and VCAM-1, on endothelial cell were determined by cell-ELISA and immunostaining assays. Moreover, mRNA and total intracellular protein expression levels of ICAM-1 and VCAM-1 were determined by real-time PCR and Western blot analysis, respectively. Keywords: saponins; Panax notoginseng; atherosclerosis; adhesion molecules

摘要 動脈粥樣硬化 (atherosclerosis)是動脈硬化的血管病中最常見而重要的一種,通 常發病人群為中年或者中老年人,現隨著我國人民生活水平提高和飲食習慣的改 變,已成為與發達國家一樣的主要死亡原因。動脈粥樣硬化主要累及體循環系統 的大型彈力型動脈(如主動脈)和中型肌彈力型動脈(以冠狀動脈和腦動脈罹患 最多),受累動脈的病變從內膜開始,先後有多種病變同時存在,包括局部脂質 和復合醣類積聚,出血和血栓形成,纖維組織增生和鈣質沉著,並有動脈中層的 逐漸退化和鈣化。現代細胞分子生物學技術顯示,動脈粥樣硬化病變都具有平滑 肌細胞增生,大量膠原纖維、彈力纖維和蛋白多醣等結締組織基質形成,以及細 胞內外脂質積聚的特點。動脈粥樣硬化一般發生在冠狀動脈,使動脈狹窄,則引 起冠心病,也可發生在主動脈、腦動脈、腎動脈等,從而引起相應部位的病變, 產生不同的嚴重後果。 目前用於降脂及抗血小板類西藥雖在一定程度上有作用,但常期服用會阻斷正常 的生理功能,具有很強的副作用。而中藥具有作用緩和且副作用小的特點,因此 我們選用我國名貴的活血化淤類中藥三七進行抗動脈粥樣硬化方面的研究。三七 為五加科(Araliaceae) 植物三七Panax notoginseng (Burk.) FH Chen. 的乾燥根。其 中,三七總皂苷為其作用的主要成分。關於三七總皂苷怎樣作用於抗動脈粥樣硬 化還沒有清晰的解說。此外,二醇型總皂苷PDS和三醇型總皂苷PTS及其它們中 主要單體Rb1和Rg1在抗動脈粥樣硬化方面的報導並不多。因此,本文將以人冠 狀動脈內皮細胞 (HCAEC)作體外模型描述三七總皂苷,二醇型總皂苷和三醇型 總皂苷及其兩個單體Rbl和Rgl是如何影響抗動脈粥樣硬化的過程。首先,用黏 附實驗觀察三七皂苷對細胞表面黏附分子的影響,再用cell-ELISA和免疫荧光檢 測PNS, PDS, PTS, Rb1和Rg1分別對細胞內黏附分子(ICAM-1) 和細胞間黏附 分子(VCAM-1) 的抑制作用。另外,在real-time PCR和Western Blotting的檢測 中,觀察粗體物和單體對ICAM-1和VCAM-1基因和蛋白表達的抑制。 結果發現,PNS, PDS,PTS,Rb1 和Rgl 均可以呈濃度依賴性地降低內皮細胞 對單核細胞的黏附作用,以PDS 的效果較為明顯:cell-ELISA 和免疫熒光的方 法檢測發現,PNS,PDS, PTS,Rb1 和 Rg1 對細胞表面的黏附分子(ICAM-1 和 VCAM-1) 有抑製作用:real-time PCR 檢測 mRNA 的表達發現三七的五個皂苷組 分都可以降低黏附分子(ICAM-1 和 VCAM-1) mRNA 的表達;Western Blotting 測定黏附分子總蛋白的表達發現五個組分對ICAM-1 的效果較為緩和,但PDS, Rb1 和Rg1 對VCAM-1 效果卻較為明顯。總體來說,三七皂苷成分對黏附分子 有抑製作用。 关键词:皂苷;三七;动脉粥样硬化;黏附分子