Backgroud: Atherosclerosis has been the number one killer for people's health. It's been proved that inflammation is the main mechanism of atherosclerosis, and the upregulation of adhesion molecule expression on vascular endothelium, such as ICAM-1, VCAM-1 and E-selectin, play an key role in the progression of inflammation. The upregulated expression of cell adhesion molecules such as ICAM-1,CAM-1 and E-selectin on the endothelial cells alters the adhesive property of the vasculature, and it is one of the key events that leads to infiltration of leukocytes across the blood vessels and hence causes inflammation. A critically regulated expression of cell adhesion molecules is therefore essential for maintaining normal homeostasis of the body. Salvia miltiorrhiza is an effective Chinese herb on prevention and treatment of cardiovascular and cerebrovascular diseases with the fuction of promoting blood circulation and removing blood stasis. Since most of studies on Salvia miltiorrhiza have mainly focused on its hydrophilic compounds, the beneficial effect of the liposoluble constituents such as Cryptotanshinoe and Tanshinone I which are comparatively more difficult to be characterized due to its poor solubility and tiny quantity, from Salvia miltiorrhiza are essentially to be explored. The study is to investigate the effect of Cryptotanshine and Tanshinone I on anti-vascular inflammation in vitro and the underlying mechanism of action. The finding of this study should provide rationales and clues for further development of Salvia miltiorrhiza on prevention and treatment of vascular inflammation associated diseases such as atherosclerosis. Obiective: To illustrate the mechanism of anti-vascular inflammatory effects of Cryptotanshine and Tanshinone I isolated from root of Salvia miltiorrhiza by determining the effect of Cryptotanshinoe and Tanshinone I on the expression of ICAM-1, VCAM-1 and E-selectin in the TNF-alpha-stimulated human umbilical vein endothelial cells (HUVEC). Methods: Firstly, the cytotoxicity of Cryptotanshinone and Tanshinone I on HUVEC cells were evaluated by XTT cell proliferation assay. Then, TNF-a induced HUVEC inflammation model was evaluated the anti-flammatory effect of the investigated compounds. The expression of ICAM-1,VCAM-1 and E-selectin were determined by cell-ELISA, immunofluorescent staining and western blot while mRNA expression of ICAM-1, VCAM-1 and E-selectin were determined by RT-PCR. Moreover, the nuclear translocation of NF-RB p65 protein was determined by immunofluorescent staining. The data was expressed as mean ‡ S.E.M. of more than three independent experiments. Results: Cryptotanshione at a concentration of higher than 10 M showed cytotoxic effect on HUVEC cells. TNF-a significantly increased the expression of ICAM-1, VCAM-1 and E-selectin in HUVEC cells ;but Cryptotanshinoe and Tanshinone I alone, below non-cytotoxic concentration, could inhibit the inducible mRNA and protein expression of ICAM-1,CAM-1 and E-selectin on the TNF-alpha activated HUVEC cells. We also found that the nuclear translocation of NF-B p65 protein was inhibited by the pre-treatment with Cryptotanshine especially at the concentration of 1 uM and 3 uM.,and Tanshinone I also inhibit the nuclear translocation of NF-B p65 protein at the concentration of 5 uM and 3 uM. Conclusions: This investigation suggested that Cryptotanshine and Tanshinone I had strong anti-inflammatory effect by inhibiting the expression of ICAM-1, VCAM-1, E-selectin and the nuclear translocation of NF-kB p65 protein, in TNF-alpha activated HUVEC cells and may play an important role in the therapeutic effect of Salvia miltiorrhiza on atherosclerosis and inflammatory responses.

研究背景：動脈粥樣硬化 atherosclerosis (AS)已經成爲現代社會危害人類健康的重大疾病之一，現代研究表明炎症是其主要發病機制，而血管內皮細胞粘附分子的表達增多是炎症的始動階段。 細胞粘附分子是一類表達於細胞表面，介導細胞與細胞之間或細胞與基質之間相互接觸與結合的糖蛋白，以配體受體相對應的形式發揮作用，在介導單核細胞與血管內膜粘附方面起重要作用。目前研究較多，作用較明確的是 ICAM-1、VCAM-1 和 E-selectin。 研究表明(在AS中ICAM-1、VCAM-1和B-selectin介導調節單核細胞的粘附及穿越内皮的遊移，使單核細胞到內皮下,攝取氧化修飾低密度脂蛋白(ox-LDL)，變爲啓動的互噬細胞，最終成爲充滿脂質的泡沫細胞，ICAM-1、VCAM-1和B-selectin還介導淋巴細胞聚集在損害部位，共同促進AS的慢性炎症過程;啓動的白細胞粘附到血管內皮能夠通過 一系列機制促進內皮細胞（EC）損傷，血管功能障礙，使 ICAM-1、VCAM-1 和B-SELECTIN等粘附分子表達進一步增加，進而叉吸引大量的白細胞，形成自我增殖的惡性循環。 丹參是在臨床應用了數千年的活血化瘀中藥，安全、有效，主要用於心腦血管疾病。現代藥理研究表明，丹參的脂溶性成分是心血管藥理活性的有效部位。隱丹參酮和丹參酮I是丹參主要脂溶性成分之一，研究表明有抗炎、殺菌、心血管保護等作用，藥理活性較強，具有很好的藥用開發價值。 目前 AS 的藥物治療，多毒副作用較大，且尚無特效藥物。因此，探討隱丹參酮和丹參酮I抗動脈粥樣硬化炎症的效果及其可能機制，對於開發安全有效的抗動脈粥樣硬化新藥，指導臨床用藥具有很強的現時意義。本課題建立了 TNF-a 誘導的人臍靜脈內皮細胞炎症模型。主要用 酸聯觅疫吸附法（ELISA），免疫螢光染色法 (Immunofluorescentstaining），蛋白質印跡法(western blot），即時螢光定量 PCR (realtime-PCR） 等方法，檢測隱丹參酮和丹參酮I對人臍靜脈內皮細胞粘附分子 ICAM-1、VCAM-1 和 E-selectin 的表達是否有抑制作用，並用 Immunofluorescent staining 法初步觀察隱丹參酮和丹參酮I對 NF-KB核轉移的作用，從而探討其抗 AS 的效果及可能的機制，爲抗動脈粥様硬化新藥的硏發提供前期硏究。 目的：探討隱丹參酮和丹參酮I對TNF-G誘導的人臍靜脈內皮細胞 (HUVEC）粘附分子ICAM-1、VCAM-1和E-selectin表達的影響，以闡明其抗動脈粥樣硬化作用及機制。爲開發安全有效的抗動脈粥樣硬化新藥進行前期研究。 方法：通過建立 TNF-Q 誘導的 HUVEC 細胞炎症模型，以抗氧化劑呲咯 烷二硫代氨基甲酸鹽（PDTC)做爲陽性對照，用ELISA、Immunofluorescent staining 測定内皮細胞表面 ICAM-1、VCAM-1 和 B-selectin 的表達，Western blot 測定內皮細胞中 ICAM-1、VCAM-1 和 B-selectin 的蛋白表達，用real time-PCR 法檢測 ICAM-1 、VCAM-1 和 E-selectin 的mRNA 表達，用 Immunofluorescent staining 檢測核轉錄因數NF-*B p65 蛋白的核轉運。 結果： TNF-@ 可顯著增加 HUVEC 細胞粘附分子 ICAM-1、VCAM-1 和 B-selectin 的表達。 隱丹參酮和丹參酮I（3MM，1HM）可明顯隆低 TNF-a 誘導的 HUVEC 細胞表面粘附分子 ICAM-1、VCAM-1 和 E-selectin 增多。 3隱丹參酮和丹參酮I(3HM，IHM)可明顯抑制TNF-Q誘導的 HUVEC 細胞 ICAM-1、VCAM-1和 E-selectin 蛋白表達、明顯抑制 TNF- a誘導的 HUVEC 細胞 ICAM-1、VCAM-1 和 E-selectin 基因表達。4 隱丹參酮和丹參酮工（3HM，1MM）對TNF-Q誘導的 HUVEC 細胞 核轉錄因數 NF-KBp65 蛋白的核轉移有抑制做用。 結論：隱丹參酮和丹參酮I可抑制TNF-Q誘導的HUVEC細胞粘附分子 ICAM-1、VCAM-1和E-selectin的蛋白與基因表達，從而抑制AS的炎症 過程，發揮抗AS的作用，可能與抑制轉錄因數NF-斤 Bp65蛋白的核轉運有關