檳榔鹼Arecoline經由TGF-β誘導人類頰黏膜纖維母細胞離氨基氧化酶樣蛋白2表現

Abstract

離氨基氧化酶樣蛋白2 (Lysyl Oxidase-like protein 2, LOXL2)是離胺基氧化酶(lysyl oxidase, LOX)家族的一種分泌型酶。主要功能是催化膠原蛋白與彈性纖維的交叉共價鍵結(cross-linking)，以維持胞外基質的硬性及穩定性。被報告和肺、肝、腎等的纖維化有關。口腔黏膜下纖維化症 (oral submucous fibrosis, OSF)，是一種不可逆的口腔黏膜纖維性病變。流行病學研究指出，嚼檳榔是口腔黏膜下纖維化症的最主要致病因子。本研究利用免疫組織化學染色方法，探討LOXL2於7例OSF及3例正常口腔黏膜(normal oral mucosa, NOM)中的表現。 結果發現在6例OSF 檢體的表皮細胞和纖維母細胞中都有LOXL2的陽性染色， NOM 則幾乎沒有表現。以檳榔鹼 arecoline 刺激人類頰黏膜纖維母細胞(buccal mucosal fibroblast, BMFs)，可以誘導BMF產生LOXL2 表現。OSF纖維母細胞與正常BMF相比，LOXL2在OSF纖維母細胞中也具有較高的表現。前處理TGF-β 中和抗體，TβRI/ALK5的專一性抑制劑SB431542，Smad3的專一性抑制劑SIS3可以抑制BMF中 arecoline誘導的LOXL2 表現。 TGF-β可以誘導BMF的LOXL2 表現。 可見得arecoline 是經由TGF-β訊息傳導路徑誘導BMF 的 LOXL2 表現。此外，前處理兒茶素(Epigallocatechin gallate, EGCG)可抑制arecoline 及TGF-β 誘導的LOXL2 表現。結果顯示EGCG對於預防或治療OSF具有極大的潛力。

Lysyl oxidase like protein 2 (LOXL2) is an extracellular copper-dependent amine oxidase that catalyses the cross-linking of collagens and elastin. It has recently been reported to serve a key role in hepatic, renal, and pulmonary fibrosis. Areca nut (AN) chewing is the most important etiological factor in the pathogenesis of oral submucous fibrosis (OSF). We examined the expression of LOXL2 protein in 7 cases of OSF and found elevated LOXL2 staining in fibroblasts and epithelial cells in 6 cases. Arecoline, a main alkaloid found in the areca nut, stimulated LOXL2 synthesis in human buccal mucosal fibroblasts (BMFs). TGFβ-neutralizing antibody, ALK5 specific inhibitor SB431542, Smad3 specific inhibitor SIS3 completely inhibited the arecoline-induced synthesis of LOXL2. Furthermore, TGF-β1 induced LOXL2 expression in BMF. Therefore, arecoline induced LOXL2 expression through TGF-β signal pathway in BMF. Moreover, Pretreated with Epigallocatechin-3-gallate (EGCG) dose-dependently inhibited arecoline-induced LOXL2 expression. In conclusion, EGCG has extreme potential for the prevention or treatment of OSF.