香菸煙霧提取物對人類臍靜脈內皮細胞鈣離子恆定及轉錄因子轉移之影響研究

流行病學的研究顯示，吸菸和心血管疾病間有強烈的相關性，吸菸直接或間接增加內皮細胞中自由基的產生並誘導發炎反應，促使白血球容易局部黏著於血管內皮細胞表面，進而造成動脈硬化症。血管內皮功能失常是動脈硬化症的早期事件，而鈣離子濃度在維持血管內皮功能也扮演重要角色。研究室先前的研究結果發現，香菸煙霧提取物 (CSE) 會對人類臍靜脈內皮細胞 (EA.hy926) 造成氧化壓力提升，誘導細胞膜上黏著分子 (ICAM-1) 增加表現，並且造成微絲骨架重組。然而，吸菸與鈣離子恆定及轉錄因子的核轉移間關係，目前仍不清楚，因此，本研究主要目的是探討CSE對人類臍靜脈內皮細胞的鈣離子恆定與轉錄因子 (Jab1、NFATc1) 核轉移影響及可能作用機制。研究結果發現，經由西方墨點法分析，24小時處理CSE造成Jab1 蛋白質總量增加；經由免疫螢光染色法分析，處理CSE 較短時間 (6或8小時)，Jab1與NFATc1在細胞核區域的表現量增加，使用鈣離子探針Fluo-3 AM偵測細胞內鈣離子濃度，發現隨著CSE處理濃度的增加，細胞內鈣離子濃度也隨之增加，CSE誘導細胞內鈣離子濃度隨著時間呈現兩階段增加。預先處理細胞內鈣離子螯合劑 (1,2-Bis (2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid tetrakis (acetoxymethyl ester), BAPTA/AM) 與細胞外鈣離子螯合劑 (Ethylene glycol tetraacetic acid, EGTA)，均可以顯著降低CSE誘導的細胞鈣離子濃度增加。另外，預處理庫藏鈣離子運轉通道抑制劑 (N-Propylargylnitrendipine, MRS 1845) 、PKC (Protein kinase C) 抑制劑GF109203X、PLC (Phospholipase C) 抑制劑U73122也發現可以有效減少CSE對胞內鈣離子濃度的增加，胞內鈣離子濃度分別為CSE處理組的70%、47%與36% (P<0.05)。綜合以上結果， CSE可能透過PLC相關路徑，調控庫藏鈣離子運轉通道 (Store-operated calcium channel, SOC) 開啟，造成細胞外鈣離子的大量流入，此鈣離子提升訊號，可能是造成相關轉錄因子Jab1和NFATc1核轉移的原因，這可能和CSE所造成的微絲骨架重組，增加血管發炎相關基因 (ICAM-1)表現有著密切關係，此一作用的釐清，在了解吸菸導致血管病變的分子機制及防治上，可以提供重要參考依據。

Epidemiologic studies have shown a strong association between cigarette smoking and cardiovascular diseases. Cigarette smoking increases directly or indirectly the production of reactive oxygen species, and induces inflammatory responses which cause leukocyte recruitment to activated endothelial cells. The dysfunctional endothelium is a major promoter for atherothrombosis and consequently cardiovascular events. Our previous results show that cigarette smoke extract (CSE) induces oxidative stress, reorganization of actin filament, oxidative stress and upregulation of ICAM-1 (intercellular adhesion molecule-1) in human umbilical vein endothelial cells (HUVEC). However, the relationship among cigarette smoking, actin filament reorganization, and calcium homeostasis, nuclear translocation of transcription factor and upregulation of inflammatory gene expression still remains unclear. Therefore, the purpose of this study is to investigate the effects of CSE on calcium homeostasis and nuclear translocation of transcription factors in EA.hy926 cells. The results showed that CSE treatment for 24 hours could partially increase total amount of Jab1 protein and their nuclear translocation detected by Western blot. Besides Jab1, CSE also leads to nuclear translocation of NFATc1 in a dose-dependent manner in EA.hy926 cells detected by immunofluorescence staining. Furthermore, CSE caused a rapid and dose-dependent increase in the intracellular free calcium level by using calcium ion sensitive dye fluo-3 as a calcium fluorescent probe. Pre-treatment with different chemical compounds, such as calcium chelators (EGTA, BAPTA AM), transient receptor potential cation channels inhibitor (MRS 1845), IP3R blocker (Xestospongin C), PKC inhibitor (GF109203X) and PLC-dependent pathway inhibitor (U73122) for 30 minutes before exposure of cells to CSE caused significant inhibition of CSE-induced calcium elevation. Moreover, the potency of efonidipine on CSE-induced elevation of intracellular calcium concentration was much less potent than other different calcium modulators used in this study. Taken together, these findings suggest that CSE-induced nuclear translocation of Jab1 and NFATc1 proteins may promote ICAM-1 expression through a calcium-dependent PKC-PLC-IP3R-mediated pathway in human endothelial cells.