Thrombin 誘導頰黏膜纖維母細胞 CTGF 表現機轉及以EGCG 治療口腔黏膜下纖維化症潛力之研究

Abstract

許多研究指出凝血酶 (thrombin) 和纖維化疾病有關。 先前研究指出結締組織生長因子 (CTGF) 也在許多纖維化疾病中扮演了重要角色。 Thrombin 可以誘導肺纖維母細胞表現CTGF。 我們先前研究發現口腔黏膜下纖維化症 (Oral submucous fibrosis，OSF) 組織中CTGF 表現量有明顯增加。檳榔鹼 arecoline 可以誘導正常人類頰黏膜纖維母細胞 (human buccal mucosal fibroblasts，BMFs)表現CTGF。 Arecoline 刺激CTGF過度表現可能是嚼食檳榔誘導OSF發生的機轉之一。 雖然OSF的致病機轉尚未完全清楚，目前認為檳榔嚼塊長期持續摩擦口腔黏膜產生微創傷合併檳榔成份引起化學刺激所導致的反應，為OSF的主要成因。 過去研究主要著重於檳榔成份引起化學刺激的反應， 雖然早期研究發現OSF病患唾液中有thrombin-like物質，微創傷引發thrombin釋出扮演的角色則未有研究。本研究發現thrombin 能以濃度與時間相關性來刺激BMFs 的CTGF表現。 Thrombin受器 (protease-activated receptors，PARs ) N端合成胜肽SFLLRN (PAR1 作用劑) 也可刺激BMFs 的CTGF表現，但PAR3, PAR4 作用劑卻無法刺激BMFs 的CTGF表現。 前處理抗氧化劑NAC、ASK-1抑制劑thioredoxin以及JNK抑制劑SP600125可以抑制Thrombin誘導的CTGF表現所抑制。 而PI3K抑制劑LY294002、 ERK抑制劑PD98059以及p38 MAPK抑制劑SB203580則不影響CTGF表現量。 BMF中thrombin透過 PAR1/ ROS/ ASK/ JNK 路徑誘導CTGF表現，可能是嚼檳榔產生微創傷誘導OSF發生的機轉之一。 另外我們亦發現綠茶酚EGCG可抑制BMF中thrombin誘導CTGF的表現。 冀望未來可以藉由抑制CTGF來達到抑制或治療口腔黏膜下纖維化症。

Thrombin has been implicated in fibrotic disorders of several organs such as kidney, liver, and lung. Connective tissue growth factor (CTGF) is associated with the onset and progression of fibrosis in many human tissues and was found to overexpress in oral submucous fibrosis (OSF). OSF is the result of persistent chemical irritation from areca nut (AN) constituents and the mechanical trauma to the oral mucosa from the coarse fibers of AN. Mechanical trauma could lead to activation of the coagulation cascade. In this study, we showed that thrombin stimulated CTGF synthesis in human buccal mucosal fibroblasts (BMFs). The effect of thrombin could be mimicked with a selective proteinase activated receptors 1 (PAR1)-activating peptide and was completely abolished with the specific thrombin serine protease inhibitor PPACK, indicating that thrombin mediated this effect via the proteolytic activation of PAR1. These results suggested that thrombin produced by microtrauma during AN chewing may contribute to the pathogenesis of OSF by up-regulating CTGF expression in BMFs. Pretreatment with antioxidant N-acetyl-L-cysteine, ASK1 inhibitor thioredoxin, JNK inhibitor SP600125, but not PI3K inhibitor LY294002, ERK inhibitor PD98059, p38 MAPK inhibitor SB203580, significantly reduced thrombin induced CTGF synthesis. Furthermore, epigallocatechin-3-gallate completely inhibited thrombin-induced CTGF synthesis. These results suggested that thrombin-induced CTGF synthesis could be mediated by reactive oxygen species, ASK1 and JNK pathways in BMFs and EGCG may serve as a useful agent in controlling OSF.