Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei, Taiwan.
Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei, Taiwan; Department of Dentistry, National Taiwan University Hospital Hsin-Chu Branch, Hsinchu, Taiwan.
J Formos Med Assoc. 2018 Jun;117(6):527-534. doi: 10.1016/j.jfma.2017.07.003. Epub 2017 Jul 15.
BACKGROUND/PURPOSE: Oral submucous fibrosis (OSF) is a premalignant condition caused by the chewing of areca nut (AN). Transforming growth factor β (TGFβ) plays a central role in the pathogenesis of OSF. Connective tissue growth factor (CTGF or CCN2) and early growth response-1 (Egr-1) are important mediators in the fibrotic response to TGFβ in several fibrotic disorders including OSF. Arecoline, a major AN alkaloid, induced the synthesis of CCN2 and Egr-1 in human buccal mucosal fibroblast (BMFs). The aims of this study were to investigate whether arecoline-induced CCN2 and Egr-1 syntheses are mediated through TGFβ1 signaling and to inspect the detailed mechanisms involved.
Western blot and TGFβ1 Emax ImmunoAssay were used to measure the effect of arecoline on the TGFβ signaling pathways. 2',7'-dichlorodihydrofluorescein diacetate and MitoSOX™ Red were used to measure the effect of arecoline on the cellular and mitochondrial reactive oxygen species (ROS).
Arecoline induced latent TGFβ1 activation, Smad2 phosphorylation, and mitochondrial and total cellular ROS in BMFs. TGFβ-neutralizing antibody completely inhibited the arecoline-induced synthesis of CCN2 and Egr-1. Mito-TEMPO, a mitochondria-targeted antioxidant, completely suppressed arecoline-induced latent TGFβ1 activation and mitochondrial and total cellular ROS. Epigallocatechin-3-gallate (EGCG) dose-dependently inhibited arecoline-induced TGFβ1 activation and mitochondrial ROS in BMFs.
Our results indicated that arecoline-induced mitochondrial ROS plays pivotal roles in the activation of latent TGFβ1 leading to the initiation of TGFβ1 signaling and subsequent increase in the synthesis of CCN2 and Egr-1. EGCG can be a useful agent in the chemoprevention and treatment of OSF.
背景/目的：口腔黏膜下纤维性变（OSF）是一种由槟榔咀嚼引起的癌前状态。转化生长因子β（TGFβ）在 OSF 的发病机制中起核心作用。结缔组织生长因子（CTGF 或 CCN2）和早期生长反应因子-1（Egr-1）是 OSF 等几种纤维化疾病中 TGFβ 诱导纤维化反应的重要介质。槟榔碱，一种主要的槟榔生物碱，诱导人颊黏膜成纤维细胞（BMFs）中 CCN2 和 Egr-1 的合成。本研究旨在探讨槟榔碱诱导的 CCN2 和 Egr-1 合成是否通过 TGFβ1 信号转导介导，并观察涉及的详细机制。
Western blot 和 TGFβ1 Emax 免疫测定用于测量槟榔碱对 TGFβ 信号通路的影响。2'，7'-二氯二氢荧光素二乙酸酯和 MitoSOX™ Red 用于测量槟榔碱对细胞和线粒体活性氧（ROS）的影响。
槟榔碱诱导 BMFs 中潜伏 TGFβ1 激活、Smad2 磷酸化以及线粒体和总细胞 ROS。TGFβ 中和抗体完全抑制了槟榔碱诱导的 CCN2 和 Egr-1 合成。线粒体靶向抗氧化剂 Mito-TEMPO 完全抑制了槟榔碱诱导的潜伏 TGFβ1 激活和线粒体及总细胞 ROS。表没食子儿茶素没食子酸酯（EGCG）剂量依赖性地抑制了槟榔碱诱导的 BMFs 中 TGFβ1 的激活和线粒体 ROS。
我们的结果表明，槟榔碱诱导的线粒体 ROS 在潜伏 TGFβ1 的激活中起关键作用，导致 TGFβ1 信号的启动，随后 CCN2 和 Egr-1 的合成增加。EGCG 可作为 OSF 化学预防和治疗的有用药物。
