State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou 510060, China.
Int J Mol Sci. 2022 May 9;23(9):5261. doi: 10.3390/ijms23095261.
Fibrosis of extraocular muscles (EOMs) is a marker of end-stage in Graves' orbitopathy (GO). To determine the antifibrotic and anti-inflammatory therapeutic effects and the underlying molecular mechanisms of disulfiram (DSF) on perimysial orbital fibroblasts (pOFs) in a GO model in vitro, primary cultures of pOFs from eight patients with GO and six subjects without GO (NG) were established. CCK-8 and EdU assays, IF, qPCR, WB, three-dimensional collagen gel contraction assays, cell scratch experiments, and ELISAs were performed. After TGF-β1 stimulation of pOFs, the proliferation rate of the GO group but not the NG group increased significantly. DSF dose-dependently inhibited the proliferation, contraction, and migration of pOFs in the GO group. Additionally, DSF dose-dependently inhibited fibrosis and extracellular matrix production markers (FN1, COL1A1, α-SMA, CTGF) at the mRNA and protein levels. Furthermore, DSF mediates antifibrotic effects on GO pOFs partially through the ERK-Snail signaling pathway. In addition, DSF attenuated HA production and suppressed inflammatory chemokine molecule expression induced by TGF-β1 in GO pOFs. In this in vitro study, we demonstrate the inhibitory effect of DSF on pOFs fibrosis in GO, HA production, and inflammation. DSF may be a potential drug candidate for preventing and treating tissue fibrosis in GO.
眼外肌纤维化（EOM）是格雷夫斯眼病（GO）终末期的一个标志。为了确定双硫仑（DSF）在体外 GO 模型中对眼外肌肌膜纤维细胞（pOF）的抗纤维化和抗炎治疗效果及其潜在的分子机制，我们从 8 名 GO 患者和 6 名非 GO 患者（NG）中建立了 pOF 的原代培养物。进行了 CCK-8 和 EdU 测定、IF、qPCR、WB、三维胶原凝胶收缩测定、细胞划痕实验和 ELISA。在 TGF-β1 刺激 pOF 后，GO 组而非 NG 组的增殖率显著增加。DSF 呈剂量依赖性抑制 GO 组 pOF 的增殖、收缩和迁移。此外，DSF 呈剂量依赖性抑制纤维化和细胞外基质产生标志物（FN1、COL1A1、α-SMA、CTGF）在 mRNA 和蛋白水平上的表达。此外，DSF 通过 ERK-Snail 信号通路介导对 GO pOF 的抗纤维化作用。此外，DSF 可减少 HA 的产生并抑制 TGF-β1 诱导的 GO pOF 中炎症趋化因子分子的表达。在这项体外研究中，我们证明了 DSF 对 GO 中 pOF 纤维化、HA 产生和炎症的抑制作用。DSF 可能是预防和治疗 GO 组织纤维化的潜在候选药物。
