| 摘要: | 抽菸是造成心血管疾病的主要原因之一。細胞微絲骨架蛋白是細胞氧化壓力早期的攻擊目標，因此可能在抽菸造成的心血管疾病中扮演關鍵性角色。實驗室先前的研究結果顯示，香菸煙霧水提取物 (cigarette smoke extract, CSE) 會快速的誘導細胞微絲骨架蛋白的重組並增加ICAM-1 (intercellular adhesion molecule-1) 在人類臍靜脈內皮細胞 (human umbilical vein endothelial cells, HUVEC) 的表現。本研究的主要目的是了解當人類臍靜脈內皮細胞 (EA.hy926) 接觸到香菸煙霧水提取物時，細胞微絲骨架所發生的變化，以及其可能之作用機制。研究結果顯示，在免疫螢光細胞染色微絲骨架下，CSE以劑量和時間依賴性方式誘導EA.hy926細胞微絲骨架發生重組，並造成細胞外形的皺縮，此外，總蛋白質羰基化 (carbonylation) 程度也隨著CSE的處理而明顯增加。將CSE與不同化合物進行共同處理細胞，包括活性含氧物種 (reactive oxygen species, ROS) 清除劑 (vitamin C、alpha-tocopherol、lipoic acid, glutathione, N-acetylcysteine)、活性含碳物種 (reactive carbonyl species, RCS ) 清除劑 (aminoguanidine)、鈣離子螯合劑 (EGTA, BAPT-AM) 及瞬態電壓感受器陽離子通道 (transient receptor potential cation channels, TRPCs) 抑制劑 (MRS 1845) 和自噬作用 (autophagy) 抑制劑 (3-methyladenine)，對受到CSE處理EA.hy926細胞的微絲骨架蛋白重組、蛋白質羰基化和細胞皺縮有不同的抑制效果。抑制CSE誘導細胞微絲骨架蛋白重組及外形皺縮的程度達到80%以上的化合物，包括硫辛酸 (lipoic acid)、穀胱甘? (glutathione)、乙二醇雙 (2-氨基乙醚) 四乙酸 (EGTA)及MRS 1845，這些結果顯示ROS的增加形成及鈣離子的恆定失調，在CSE造成微絲骨架重組及外形皺縮上扮演重要的角色。除此之外，本研究以免疫螢光觀察胞內自噬作用 (autophagy) 標記LC3蛋白 (microtubule-associated protein 1 light chain 3），發現CSE處理也明顯造成LC3蛋白的分佈集中於細胞的皮層 (cortex)，整體表現量似也有增加的情形，這項發現指出CSE處理可能干擾LC3蛋白的易位 (translocation)並影響自嗜作用的進行，因而增加細胞的死亡。
Cigarette smoking is a major cause of cardiovascular disorders. Actin cytoskeleton is a possible key player in responding to inflammatory stimuli and also an early target of cellular oxidative stress. Laboratory previous results also show that cigarette smoke extract (CSE) induces rapid actin cytoskeleton remodeling and up-regulate ICAM-1 (intercellular adhesion molecule-1) expression in human umbilical vein endothelial cells (HUVEC). The purpose of this study is to understand the actin damages and its possible mechanism in human umbilical vein endothelial cells EA.hy926 exposed to cigarette smoke extract (CSE). The results showed that CSE caused dose- and time-dependent reorganization of actin cytoskeleton and cell shrinkage in EA.hy926 cells by staining with rhodamine phalloidin. In addition, CSE increased total protein carbonylation in a dose-dependent manner. Cells co-treated with different chemical compounds, such as reactive oxygen species scavenger (vitamin C, alpha-tocopherol, lipoic acid, glutathione, N-acetylcysteine), reactive carbonyl scavengers (aminoguanidine), calcium chelator (ethylene glycol tetraacetic acid, BAPTA-AM ), transient receptor potential cation channels inhibitor (MRS 1845), and autopgay inhibitor (3-methyladenine) with CSE caused different inhibition of actin reorganization, protein carbonylation, and cell shrinkage in CSE-treated EA.hy926 cells. Among these compounds, lipoic acid, glutathione, EGTA, and MRS 1845 were found to potently inhibit the CSE-induced actin cytoskeleton reorganization and cell shrinkage. Furthermore, immunofluorescence staining analysis showed that the autophagy marker LC3 protein (microtubule-associated protein 1 light chain 3) accumulated at cell cortex in CSE-treated cells. Collectively, these findings suggest that CSE-mediated ROS generation and Ca2+ influx increase through TRPC channels are the key factors that may cause reorganization of actin cytoskeleton and the subsequent cell shrinkage. In addition, CSE-induced inappropriate organization of the actin cytoskeleton may dysregulate autophagy by abnormal accumulation of LC3 proteins at cell cortex. |
