抽菸除造成肺癌、慢性肺阻塞及其他肺部疾病外，對心血管系統也造成相當大的危害， 抽菸造成癌症及心血管系統疾病的研究雖然很多，但到目前仍沒有清楚明確的解答，最 主要的因素與香菸組成分複雜有著密切關係，香菸煙霧中含近 7000 種不同的成分，此 一複雜情形，唯有透過持續性的研究或可在特定區塊提供較為完整明確的解釋。本研究 室最近的試驗發現：香菸煙霧水提取物(cigarette smoke extract/CSE)在人類血管內 皮 EA.hy926/HUVEC 細胞，可依 CSE 處理的濃度及時間，明顯造成細胞內鈣離子濃度的 增加，以及微絲骨架的重組和細胞外形皺縮，進一步更此現象可被蛋白激酶 C (protein kinase C/PKC)抑制劑 GF109203X、磷脂酰肌醇磷脂酶 C (phosphatidylinositol phospholipase C/PLC)抑制劑 U73122 及瞬時受體電位陽離子通道(transient receptor potential cation channel/TRPC)抑制劑活 MRS 1845 有效的壓制(block)，這些結果明 顯指出 PLC-PKC-TRP 途徑，在 CSE 造成人類血管內皮細胞鈣離子濃度增加，以及微絲骨 架重組扮演極重要的調節角色。為進一步探討此控制途徑的作用細節，並且評估此現象 在防治菸害可能之應用價值，本研究計畫將以二年時間，利用人類血管內皮 (EA.hy926/HUVEC)細胞為模式，進行何種特定 PKC 及 PLC 同功酶(isoenzyme)參與 CSE 在人類血管內皮細胞造成鈣離子濃度增加及微絲骨架重組的確定，為第一年研究之主 軸；第二年則以探討維持微絲骨架動態恆定相關蛋白質，在 CSE 造成 EA.hy926/HUVEC 細胞鈣離子濃度增加及微絲骨架重組所扮演的角色為主軸，這些蛋白質包括(1)微絲切 割蛋白「gelsolin、villin 及 scinderin」及(2)微絲結合蛋白「MARCKS (Myristoylated alanine-rich C-kinase substrate)、IQGAP1(IQ motif containing GTPase activating protein 1)及 filamin A」。同時，本研究計畫也將利用鈣離子濃度增加及微絲骨架重組 兩種參數，評估保護肺臟名方-『百合固金湯』中藥組成之防菸害活性，來加強本研究 之應用價值。內皮屏藏障(endothelium barrier)的完整性缺損是血管發炎，形成動脈 粥狀硬化及肺性高血壓重要的原因，與抽菸造成的心血管病有著重要關連，因此，本研 究計畫成果，對未來在抽菸造成發炎及心血病防治議題上，可提供基礎及應用之建議。 Cigarette smoking not only induces lung cancer, chronic obstructive pulmonary disease (COPD) and other lung diseases, but also causes cardiovascular diseases. Although there have been lots of studies on the negative effects of cigarette smoking, the exact molecular mechanism of cigarette smoking-induced cardiovascular diseases is still unclear. One of the major reasons is its complicated composition of cigarertte smoke. More than 7,000 different compounds have been found in cigarettes smoke. Our recent results showed that cigarette smoke extract induced significant elevation of intracellular calcium ion concentration as well as the reorganization of microfilament cytoskeleton in a dose- and time-dependent manner in human endothelail EA.hy926 cells. Furthermore, these phenomena could be potently suppressed by protein kinase c inhibitor GF109203X, phosphoinositide phospholipase C inhibitor U73122 and transient receptor potential cation channel inhibitor MRS 1845. These results clearly indicate that PLC/PKC/TRPC signaling pathway plays an extremely important role in the CSE-induced intracellular calcium concentration increase and microfilament reorganization in EA.hy926 cells. To further uncover the detail mechanism of this possible signaling pathway, this two-year research project will use human endothelial cell lines (EA.hy926 or HUVEC) as a model system to clarify the following two important questions: (1) which specific isoensyme of PKC and PLC is involved in CSE-induced calcium ion concentration increase? (2) What is the role of actin-severing proteins (gelsolin, villin and scinderin) and actin-binding proteins [MARCKS (Myristoylated alanine-rich C-kinase substrate), IQGAP1 Filamin A] on CSE-induced microfilament reorganization? At the same time, this research project will also use CSE-induced elevation of calcium ion concentration and reorganization of microfilament cytoskeleton as screening parameters to evaluate the anti-cigarette smoking potential of the drugs that is composed of a famous Chinese traditional medicine formula named “Bae-Her-Guh-Jin-Tang (百合固金湯)” for preventing lung from damage. The defect of endothelium barrier integrity is a key reason for developing of blood vessels inflammation and pulmonary hypertension and it has a significant correlation with cigarette smoking-related cardiovascular diseases. The results of this study can provide the basic molecular mechanism of how CSE induced elevation of calcium ion conentration and reorganization of microfilament, and explore the possible drug(s) as TRPC inhibitor to prevent the cells against CSE-induced elevation of calcium ion concentration in human endothelial cell lines.
