- 學位論文
沒食子酸誘導小鼠肺纖維母細胞凋亡之分子作用機轉
Molecular mechanism of gallic acid-induced apoptosis in primary cultured mouse lung fibroblasts
摘要
肺纖維化是因肺間質組織過度累積細胞外間質，如：膠原蛋白，所造成之病症。多種慢性發炎之肺間質性疾病皆會使肺纖維母細胞活化，使膠原蛋白不斷分泌，造成肺纖維化，而其中詳細相關的機轉至今仍尚未被瞭解。目前針對肺纖維化之治療，尚無一確切之治療策略，僅能以藥物抑制或透過外科手術治療，但卻都會造成一些不良的副作用，反而影響癒後結果。因此，找尋新的藥物提高肺纖維化病患之存活率是十分重要的醫藥研發方向。近年來西方醫學界廣泛的利用中草藥或由植物中萃取有效成分來治療疾病，其中具有特定藥效，能有效的治療或防治一些西方醫藥所不能控制的疾病。沒食子酸（Gallic acid），為單寧酸的一種，存在於許多植物中，研究證實其具有抗菌、抗氧化、抗發炎及抗腫瘤等功效。本研究，探討沒食子酸對肺纖維母細胞生長存活之影響並研究其作用機制，以評估其抗纖維化的效果。由細胞生長曲線及流式細胞儀的分析的結果，發現沒食子酸呈劑量及時間依存性的使細胞停留在sub-G1 期，並造成細胞死亡。由染色結果得知，其為誘導細胞凋亡；其中caspase的活化扮演著重要角色。沒食子酸會誘導肺纖維母細胞中產生ROS ，造成粒線體膜電位下降；若前處理抗氧化劑Ascorbic acid ，可抑制沒食子酸產生ROS所造成的細胞凋亡。西方墨點法之結果得知，沒食子酸促使肺纖維母細胞凋亡，其分子機轉是透過死亡受體路徑的活化所造成。本研究結果顯示，沒食子酸為一極具潛力治療肺纖維化的天然化合物。
並列摘要
Lung fibrosis refers to the scars characterized by the accumulation of large ECM such as collagens through the whole lung. It is well known that fibrosis is caused by the chronic and permanent activation of the fibroblast which induced by immune inflammation response; but whose mechanism is poorly understood. Thus, lots of efforts had been made to pursuit of the new drug capable of reversing/ blocking fibroblast activation. In this study, by using gallic acid, which is a component of tannin found in plants, we successfully demonstrated its rapid killing effects toward mice primary lung fibroblast in both time and dosage-dependent manners; the following results showed that gallic acid is mainly to induce mice primary lung fibroblast apoptosis. Besides, we found that the addition of gallic acid caused the disruption of mitochondrial membrane potential. In addition, the results also showed that gallic acid induced the activation of caspase-2 and -3 and enhanced the expression levels of Fas, Fas ligand, p-AKTser473 , p-AKTser308 and p-ERK, but not the levels of Bcl-2 family molecules and TRAIL （Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand） death receptor ; suggesting a positive/promoting correlation between gallic acid-induced mice lung fibroblast apoptosis and the increasing expression pattern of Fas receptor and caspase-3 and -8 activations. The results indicated that gallic acid might be worthy to be elucidated further to dissect its therapeutic role in anti-lung fibrosis process.