| 摘要: | 尼古丁已被廣泛證明為心血管病變如動脈粥樣硬化(Atherosclerosis)主要的危險因素之一。然而，在先前的研究中缺少了一項影響人類血管內皮生理的重要因子：層流剪應力(Laminar Shear Stress)，因此尼古丁在實際血管內如何導致內皮細胞傷害的機制是有待證實的。在本研究中，我們利用人類臍靜脈內皮細胞(Human umbilical vein endothelial cells; HUVECs)作為實驗細胞，施以尼古丁做藥物處理，並結合平行板流道系統模擬層流剪應力，進而來探討血流動力學在尼古丁介導的人類內皮細胞發炎情況的影響。首先HUVECs會以12 dynes/cm2在±10-4M尼古丁的環境下以不同的時間沖刷：時間設定為0(靜態組), 6, 12, 24 小時，接著以血球貼附實驗、活性氧自由基測量，以及西方墨點法分析細胞貼附因子表現等方法來說明內皮細胞的發炎情形。實驗結果顯示，HUVECs在沒有尼古丁的環境下，層流剪應力在24小時內對血球貼附率沒有顯著影響(P > 0.05)；而HUVECs在沒有層流剪應力(靜態)的環境下，尼古丁在12小時以及24小時分別提升了血球貼附率1.8倍(P < 0.05)以及2.6倍(P < 0.05)。然而，在結合尼古丁以及層流剪應力處理24小時的組別中，血球貼附率會比以單獨尼古丁處理24小時的組別高1.4倍(P < 0.05)。而此結果與活性氧自由基測量的結果是呈現一致的；在結合尼古丁以及層流剪應力處理24小時的組別中，細胞產生的活性氧會分別比以單獨尼古丁處理24小時或單獨以層流剪應力處理24小時的組別提升2.55倍(P < 0.05)以及4.22倍(P < 0.05)。接著我們以Intercellular Adhesion Molecule-1 (ICAM-1)以及Vascular Adhesion Molecule-1 (VCAM-1)兩種貼附因子蛋白作西方墨點法實驗，分析內皮細胞在尼古丁以及層流剪應力處理後的表現，我們發現ICAM-1以及VCAM-1的表現在單獨尼古丁處理24小時的情況下會分別提升1.3倍(P < 0.05)以及1.9倍(P < 0.05)；而在單獨以層流剪應力處理24小時的情況下ICAM-1以及VCAM-1的表現並無顯著的提升(P > 0.05)；然而，尼古丁結合層流剪應力則會使ICAM-1表現比單獨尼古丁處理情況下提升1.6倍(P < 0.05)，不過卻會使VCAM-1表現下降0.9倍(P > 0.05)，此結果顯示了在有尼古丁環境下，層流剪應力在調節內皮細胞上ICAM-1以及VCAM-1的表現扮演了不一樣的角色。本研究顯示了內皮細胞在同時受到尼古丁與層流剪應力刺激24小時的發炎程度會比單獨受到尼古丁或是層流剪應力刺激有顯著的提升(P值均小於0.05)，此一結果顯示了層流剪應力在尼古丁造成的內皮細胞發炎反應上扮演了一個不利的角色。;Nicotine has been widely demonstrated as one of major risk factors for cardiovascular lesions such as atherosclerosis. However, the definite mechanism of how nicotine causes endothelial dysfunction remains unclear due to lack of hemodynamic factor in most of prior in vitro studies. In this study, we aimed to investigate the effectiveness of hemodynamic stress on nicotine-mediated human endothelial inflammation using human umbilical vein endothelial cells (HUVECs) and parallel-plate flow system to simulated blood flow-induced laminar shear stress (LSS). HUVECs were first sheared by 12 dynes/cm2 using medium with and without 10-4 M nicotine for 0 (static culture), 6, 12, and 24 h, respectively, and followed by conductions of monocyte adhesion assay, detection of reactive oxygen species (ROS) generated, and analysis of inflammation-related molecular expression. Our results showed that the monocyte adhesion rate (MDR), which was represented as the level of cell inflammation, was not affected by LSS within 24 h in the absence of nicotine. As compared to the shearing cultures without nicotine, the MDR of the group treated by both nicotine and LSS remarkably increased about 1.8- and 2.6-fold (P < 0.05 for each) while the exposure time was set at 12 and 24 h, respectively. Moreover, the inflammation level of HUVECs treated with both LSS and nicotine for 24 h significantly increased about 1.4 fold (P < 0.05) as compared to the group with nicotine alone. These results were consistent with the data obtained from the ROS measurement that the level of ROS generated from the cells with both LSS and nicotine exhibited 2.55- and 4.22-fold (P < 0.05 for each) higher than the groups with nicotine or LSS alone, respectively. To investigate the mechanism of enhanced endothelial inflammation caused by synergistic effect of LSS and nicotine, expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) were examined. We found that expressions of both ICAM-1 and VCAM-1 increased 1.3 and 1.9-fold while cells were exposed to 10-4 M nicotine for 24 h, whereas LSS enabled to further increased the ICAM-1 but decreased the VCAM-1 expression of nicotine-treated HUVECs after sheared with 12 dynes/cm2 for 24 h, This result suggested that LSS plays different roles in modulating ICAM-1 and VCAM-1 expressions in endothelial cells in the presence of nicotine. In summary, we have demonstrated that the level of endothelial inflammation can be significantly increased by synergistic effect of nicotine and LSS, and exhibited that high level of LSS (≥ 12 dynes/cm2) plays a detrimental role in nicotine-mediated endothelial injury. |
