| 摘要: | Cigarette smoking is a major cause of cardiovascular
disorders. Smoking-induced inflammation and other
risk factors like dyslipidemia cause vascular
endothelial damage via oxidative stress. The role of
the actin cytoskeleton as a possible key player in
the response to inflammatory stimuli and it is an
early target of cellular oxidative stress. Our
previous results also show that cigarette smoke
extract (CSE) induces rapid actin cytoskeleton
remodeling and up-regulate ICAM-1 expression in Human
Umbilical Vein Endothelial Cells（HUVE. The purpose
of this research project is to understand how CSE
induced pro-inflammation gene ICAM-1 upregulation in
human endothelial EA.hy926 cells. Fluorescence
staining of EAhy926 cells incubated with CSE showed
that organization of the actin cytoskeleton was
disrupted and caused cell to shrink in a dose- and
time-dependent manner. By means of immunofluorescence
analysis technique, CSE-induced EA.hy926 cells actin
reorganization, cell shrinkage was increased with a
dose and time course manner. In addition, actin
cytoskeleton reorganization, proteincarbonylation and
cell shrinkage extent was significantly induced by
CSE exposure. Pretreatment of antioxidants (lipoic
acid, glutathione peptide, N- acetyl cysteine,
aminoguanidine, a-tocopherol, vitamins C), calcium
ion chelates and calcium channel inhibitors (EGTA,
BAPTA AM, MRS 1845) and autophagy inhibitor (3-MA)
could obviously block CSE-induced protein
carbonylation, actin reorganization and cell
shrinkage. The inhibitory activity from high to low
order is: LA, GSH, EGTA, MRS, NAC > AG, TCP, BAPTA-AM
> 3-MA, VitC. These results showed that CSE -induced
actin reorganization mechanism, at least contains
intracellular ROS production and a rise in
intracellular calcium ions concentration. Similar
situation, CSE-induced protein carbonylation of
endothelial cells and the up-regulation of proinflammation
gene, ICAM-1 could also be significantly
attenuate by antioxidants and calcium ion chelators
or calcium channel inhibitor. In addition, using
immunofluorescence microscope technique also found
that CSE can significantly increase AP-1 co-activator
Jab1 protein nuclear translocation. Results from this
study not only provide a basic explanation of the
mechanism of how CSE-induced cardiovascular disorders
but also offer practical suggestions to
antioxidant/calcium regulators therapy for CSEmediated
diseases prevention.
抽菸與心血管疾病形成之間有著密切的關係。抽菸所引起的
發炎反應和其他危險的因子例如高血脂等的交叉作用，將增
加血管內皮細胞的氧化壓力，進而引起血管內皮細胞損傷，
最終造成嚴重的血管病變，例如動脈粥狀硬化症。本研究室
先前的研究結果顯示，香菸煙霧水提取物（cigarette smoke
extract，CSE）在人臍靜脈內皮細胞（human umbilical
vein endothelial cells，HUVEC）有誘導蛋白質氧化羰基
化、細胞微絲骨架蛋白重組，以及增加ICAM-1 蛋白質的表現
程度。本研究計畫目的是進一步了解人類臍靜脈內皮細胞
(EA.hy926)處理香菸煙霧水提取物的時，細胞微絲骨架是如
何發生重組變化，進而增加ICAM-1 蛋白質的表現。藉由細胞
螢光骨架染色分析，證明CSE 誘導EA.hy926 細胞微絲骨架的
重組和所造成細胞的皺縮，有隨劑量增加和時間增長而提高
的現象。此外，蛋白質羰基化的程度也隨著CSE 處理濃度的
增加而明顯上升，預處理抗氧化劑（硫辛酸、穀胱甘肽、N-
乙酰半胱氨酸、氨基胍、tocopherol、維生素C）、鈣離子
螯合劑及鈣離子通道抑制劑(乙二醇四乙酸、BAPTA AM、MRS
1845)及細胞自噬作用抑制劑(3-MA)，均有不同程度減緩CSE
所誘導的細胞微絲骨架蛋白重組、細胞皺縮和蛋白質羰基化
的程度，抑制活性由高至低依序為：LA、GSH、EGTA、MRS、
NAC > AG、TCP、BAPTA-AM > 3-MA、VitC。此外藉由免疫螢
光觀察light chain 3（LC3），也發現CSE 處理會提高細胞
內自噬作用標籤LC3 的表現，預處理抗氧化劑CSE 造成之
LC3 螢光量也隨之下降。這些結果顯誓，CSE 所誘導的微絲骨
架重組的機制中，至少包含細胞內ROS 產生增加，以及細胞
鈣離子濃度上升兩種訊息。相似的狀況，CSE 所造成內皮細
胞蛋白質羰基化及發炎前驅基因ICAM-1 增加表現，也可被抗
氧化劑及鈣離子螯合劑或鈣離子通道抑制劑的添加而明顯抑
制。另外，以免疫螢光顯微鏡技術觀察，更發現CSE 處理會
明顯增加AP-1 輔助活化劑Jab1 蛋白質的細胞核轉移
(nuclear translocation)，綜合上述所有的數據，我們成功
證實CSE 誘發血管內皮細胞ICAM-1 增加表現的分子機制，包
含下列數個階段。階段一：促進細胞內ROS 上升；階段二：
形成ER stress 並釋放鈣離子；階段三：細胞外大量鈣離子
流入；階段四：促進蛋白質大量的氧化羰基化並釋放轉錄因
子Jab1 蛋白質；階段五：藉微絲骨架重組，促成Jab1 蛋白
質的細胞核轉移；階段六：Jab1 蛋白質輔助c-Jun 或AP-1
並增加其轉錄活性，最終增加ICAM-1 基因的表現。本研究成
果對抽菸如何導致動心血管疾病可提供解釋之基礎，對利用
抗氧化劑或鈣離子抑制劑來防治菸害也提供重要的參考依
據。 |
