EGCG抑制檳榔鹼Arecoline經由TGF-β1訊息傳遞路徑誘導人類頰黏膜纖維母細胞及上皮細胞早期生長反應因子表現之研究

Abstract

嚼食檳榔是造成口腔黏膜下纖維化症（Oral submucous fibrosis，OSF）的最主要原因。文獻指出檳榔中的主要成分檳榔鹼（Arecoline）會誘導許多纖維化相關基因的表現。早期生長反應因子（Early growth response-1，Egr-1）是一個轉錄因子，研究發現Egr-1的表現與纖維化疾病呈現高度正相關性，但Egr-1在OSF中扮演角色並不清楚。本研究首先利用免疫組織化學染色方法，發現在正常口腔黏膜（NOM）中，其上皮細胞並沒有Egr-1的表現，在頰黏膜纖維母細胞（buccal mucosal fibroblast，BMF）及發炎細胞的細胞質與細胞核中，則有發現些微的Egr-1表現。在OSF組織切片中，其上皮細胞、BMF細胞及發炎細胞的細胞質與細胞核皆有Egr-1的陽性濃染。接著我們發現Arecoline會誘導BMF細胞中Egr-1的表現，而Src抑制劑、ERK抑制劑、JNK抑制劑以及抗氧化劑NAC能夠明顯降低Arecoline誘導BMF細胞中Egr-1的表現。此外，我們發現兒茶素（EGCG）對於Arecoline誘導BMF細胞中Egr-1的表現、collagen的合成以及肌纖維母細胞的分化皆有明顯的抑制效果。此外，Src抑制劑、ERK抑制劑、JNK抑制劑、ALK5抑制劑、Smad3抑制劑以及Smad3 siRNA能夠明顯降低TGF-β1誘導BMF細胞中Egr-1的表現。Egr-1 siRNA可以明顯抑制TGF-β1誘導BMF細胞collagen的合成以及肌纖維母細胞的分化。此外，EGCG可以抑制TGF-β1誘導BMF細胞中Egr-1的表現、collagen的合成以及肌纖維母細胞的分化。進一步發現TGF-β1會誘導BMF細胞中NADPH oxidase 4（NOX4）的表現，而抗氧化劑NAC，NOX family抑制劑以及NOX4抑制劑能夠抑制TGF-β1誘導BMF細胞中Egr-1的表現及ROS的產生。抗氧化劑NAC能夠抑制TGF-β1誘導BMF細胞中Src的磷酸化，NOX4抑制劑、Src抑制劑及NOX4 siRNA可以有效抑制TGF-β1誘導BMF細胞中Src、ERK、JNK及Smad3的磷酸化，顯示NOX4/ROS位於Src上游且Src位於ERK、JNK及Smad3的上游。我們使用TGF-β中和抗體、ALK5抑制劑以及Smad3抑制劑發現可有效降低Arecoline誘導BMF細胞中Egr-1的表現、膠原蛋白的合成以及肌纖維母細胞的分化，顯示Arecoline會開啟TGF-β訊息傳遞路徑。進一步發現Arecoline可以增加BMF細胞培養液中活化態TGF-β1的表現量。前處理抗氧化劑NAC及GSH能夠明顯降低Arecoline誘導BMF細胞的活化態TGF-β1。抗氧化劑NAC及Trolox，PEG-catalase，mitochondria-targeted O2-抑制劑以及MnTBAP能夠明顯抑制Arecoline誘導BMF細胞活化態TGF-β1的表現量。直接加入H2O2會誘導BMF細胞活化態TGF-β1的表現，且mitochondria-targeted O2-抑制劑可以明顯降低Arecoline誘導BMF細胞mitochondrial superoxide的產生。這些結果顯示Arecoline是經由粒線體來源的Superoxide（O2-）及H2O2來誘導BMF細胞活化TGF-β1。EGCG可抑制Arecoline誘導BMF細胞mitochondrial superoxide的產生以及TGF-β1的活化。過去研究發現，口腔上皮細胞受到TGF-β刺激會產生上皮-間質細胞轉化 (Epithelium-mesenchymal transition，EMT)，在OSF的進程中扮演重要角色。我們選用頰黏膜上皮細胞株TW2.6細胞來探討Egr-1是否扮演調控EMT的角色。我們發現TGF-β1會誘導TW2.6細胞中Egr-1及Vimentin的表現，並降低E-cadherin的表現。使用Egr-1 siRNA可以明顯抑制TGF-β1誘導TW2.6細胞中Egr-1及Snail的表現，並且回復EMT的現象。我們發現Src抑制劑、JNK抑制劑、抗氧化劑NAC、NOX family抑制劑、NOX4抑制劑、ALK5抑制劑以及Smad3抑制劑皆可明顯抑制TGF-β1誘導TW2.6細胞中Egr-1及Snail的表現。我們還發現EGCG可以明顯抑制TGF-β1誘導TW2.6細胞中Egr-1及Snail的表現，並且回復EMT的現象。這些結果顯示EGCG對於預防或治療OSF具有極大的潛力。

Areca nut (AN) chewing is the most important risk factor of oral submucous fibrosis (OSF). Early growth response-1 (Egr-1) protein has been shown to play a central role in the pathogenesis of many human fibrotic diseases. We immunohistochemically examined the expression of Egr-1 protein in OSF specimens. Positive Egr-1 staining was detected in subepithelial fibroblasts and inflammatory cells. We further observed Arecoline, a main alkaloid found in AN, increased Egr-1 synthesis in a dose- and time- dependent manner in buccal mucosal fibroblasts. Constitutive overexpression of Egr-1 during AN chewing may play a role in the pathogenesis of OSF. Pretreatment with antioxidant NAC, JNK inhibitor and ERK inhibitor significantly reduced Arecoline induced Egr-1 synthesis. Furthermore, epigallocatechin-3-gallate (EGCG) completely inhibited Arecoline-induced Egr-1 synthesis and the inhibition is dose-dependent. Transforming growth factor β (TGF-β) is a key regulator in the pathogenesis of OSF. Egr-1 is an important mediator of TGF-β-induced responses and is considered to play a central role in orchestrating fibrotic responses. Egr-1 may contribute to the pathogenesis of OSF as it is overexpressed in OSF specimens and in Arecoline-stimulated normal human buccal mucosal fibroblasts (BMFs). Previous study has shown that the stimulation of Egr-1 by TGF- in dermal fibroblasts occurs independent of the activin receptor-like kinase 5 (ALK5)/Smad3 signaling axis. Since Egr-1 is essential for the fibrotic response to TGF- and that TGF- signaling is cell type-and context-dependent, we aimed to examine the possible signal transduction pathways involved in TGF-β1-induced Egr-1 expression in BMFs. In the present study, we have shown that TGF-β1 induces Egr-1 expression and that Egr-1 mediates the TGF-β1-induced mRNAs expression of the α1- and α2-chains of type I collagen (COL1A1 and COL1A2) and the acid-soluble collagen production in BMFs. We have also shown that ALK5 inhibitor, ERK inhibitor, JNK inhibitor, and Smad3 inhibitor significantly abrogate the TGF-β1-induced levels of Egr-1 protein, indicating that ALK5/Smad3, ERK, and JNK are involved in the TGF-β1-induced Egr-1 in BMFs. We have further demonstrated that knocking down Smad3 using Smad2/3 siRNA completely abolishes the TGF1-induced Egr-1. Moreover, EGCG fully inhibits TGF-β1-induced Egr-1 expression and abrogates the TGF-1-stimulated production of acid-soluble collagens. We found TGF-β1 induced NADPH oxidase 4 (NOX4) expression in BMFs. Pretreatment with antioxidant NAC, NOX family inhibitor, and NOX4 inhibitor significantly reduced TGF-β1-induced Egr-1 expression and ROS production in BMFs. We found antioxidant NAC significantly inhibited TGF-β1-induced Src phosphorylation in BMFs. In addition, NOX4 inhibitor, Src inhibitor, and NOX4 siRNA significantly inhibited TGF-β1-induced Src, ERK, JNK, and Smad3 phosphorylation in BMFs. These results showed that TGF-β1-induced Src phosphorylation is regulated by NOX4/ROS, and Src is an upstream signaling transducer of ERK, JNK, and Smad3 in BMFs. We found Arecoline increased activated TGF-β1 level in BMFs. Pretreatment with antioxidant NAC and GSH significantly reduced Arecoline-induced activated TGF-β1 level in BMFs. We further found antioxidant NAC and Trolox, PEG-catalase, mitochondria-targeted O2- inhibitor, and MnTBAP significantly inhibited Arecoline-induced activated TGF-β1 level in BMFs. We found H2O2 increased activated TGF-β1 level in BMFs. Mitochondria-targeted O2- inhibitor significantly reduced Arecoline-induced mitochondrial superoxide in BMFs. These results showed that Arecoline increased activated TGF-β1 level via mitochondrial superoxide and H2O2 in BMFs. We further found EGCG significantly inhibited Arecoline-induced mitochondrial superoxide and activated TGF-β1 level in BMFs. Epithelial-mesenchymal transition (EMT) is a big issue in fibrosis disease. We investigate whether Egr-1 play a role in regulating EMT in TW2.6 cells. We found TGF-β1 induced Egr-1 and Vimentin expression and reduced E-cadherin expression in TW2.6 cells. Genetic targeting of Egr-1 by Egr-1 siRNA significantly inhibited TGF-β1-induced Egr-1 and Snail expression and reduced TGF-β1-induced EMT in TW2.6 cells. We found Src inhibitor, JNK inhibitor, antioxidant NAC, NOX family inhibitor, NOX4 inhibitor, ALK5 inhibitor, and Smad3 inhibitor significantly inhibited TGF-β1-induced Egr-1 and Snail expression in TW2.6 cells. We further found EGCG significantly inhibited TGF-β1-induced Egr-1 and Snail expression and reduced TGF-β1-induced EMT in TW2.6 cells. EGCG potentially qualifies as a useful reagent for the prevention and therapy of OSF.