凝血酶經由活化TGF-β1誘導人類頰黏膜纖維母細胞結締組織生長因子表現之研究

Abstract

口腔黏膜下纖維化症 (Oral submucous fibrosis, OSF) 是一種慢性發炎的口腔黏膜病變，臨床症狀為口腔黏膜僵硬、張口、吞嚥以及說話困難。過去研究研究發現嚼食檳榔與 OSF 的產生密切相關。檳榔鹼 (arecoline)、變形生長因子β (TGF-β1)、結締組織生長因子 (CCN2)、微創傷在OSF致病機轉扮演重要角色。先前本實驗室發現，凝血酶在口腔頰黏膜纖維母細胞 (human buccal mucosal fibroblasts, BMFs) 可透過 PAR1、ROS、ASK1、JNK誘導 CCN2 表現；而 TGF-β1 則透過 ALK5、JNK 或 p38 路徑誘導 CCN2 表現，而兩者的 CCN2 表現皆可被茶多酚 (EGCG) 抑制。本研究進一步探討在 BMFs 中 Thrombin 是否會經由TGF-β1誘導頰黏膜纖維母細胞 CCN2 表現及其機轉。 我們首先發現Thrombin可誘導BMFs p-Smad3 的表現。進一步發現TGF-β1 中和抗體、ALK5抑制劑 SB431542、Smad3 抑制劑 SIS3可抑制 Thrombin 誘導的 CCN2 表現，表示 Thrombin 誘導 CCN2 的表現路徑中間有 TGF-β1 訊息路徑的參與。前處理 RGD blocking-peptide 和 integrin 中和抗體亦可以抑制 Thrombin 誘導的活化態 TGF-β1、p-Smad3、以及 CCN2 的表現，顯示 Thrombin 活化 TGF-β1 訊息路徑的過程中有 integrin 的參與；而前處理 ROCK 抑制劑 Y27632、Actin 聚合抑制劑 Blebbistatin 都可以抑制 Thrombin 誘導的活化態 TGF-β1、p-Smad3、以及 CCN2 的表現。顯示在 BMFs 中，Thrombin 可能透過 PAR1/ROCK/Actin polymerization/integrin 訊息路徑調控 TGF-β1 訊息路徑來誘導 CCN2 表現。另外我們亦發現 EGCG 可抑制 Thrombin 誘導的活化態 TGF-β1。EGCG可藉由抑制活化態 TGF-β1來達到抑制或治療口腔黏膜下纖維化症。

Oral submucous fibrosis (OSF) is a chronic inflammatory disease. Its clinical sign and symptoms include stiffness of oral mucosa, difficulty in mouth opening, swallowing and speech. The regular use of areca nut (AN) is the major etiological factor of OSF. Previous study indicated that arecoline, TGF-β1 and microtrauma play an important role in the pathogenesis of OSF. We previously showed TGF-β1 induced CCN2 through ALK5/JNK p38 pathway in human buccal mucosal fibroblasts (BMFs). Thrombin induced CCN2 expression through PAR1/ROS/ASK1/JNK pathway in BMFs. Both of them can be inhibited by epigallocatechin-3-gallate (EGCG). The aim of this study was to investigate whether thrombin induced CCN2 expression in BMFs is via TGF-β1 activation. We first found thrombin induced Smad3 activation in BMFs. Pretreatment with TGF-β1 neutralizing antibody, ALK5 inhibitor SB431542 and Smad3 inhibitor SIS3 significantly reduced thrombin induced CCN2 expression. These results indicate that thrombin-induced CCN2 expression in BMFs is via TGF-β1-dependent pathway. Pretreatment with RGD blocking-peptide and integrin neutralizing antibodies significantly reduced thrombin induced activated TGF-β1 level, p-Smad3 and CCN2, suggest that integrin is involved in thrombin activate TGF-β1 pathway. Furthermore, ROCK inhibitor Y27632, actin/myosin destabilizing agent blebbistatin significantly reduced thrombin induced activated TGF-β1 level, p-Smad3 and CCN2. In addition, EGCG completely inhibited thrombin-induced activated TGF-β1 level. These results indicated that thrombin-induced CCN2 expression is via TGF-β1 pathway and mediated by PAR1/ROCK/Actin polymerization/integrin cascade.