Disulfiram Exerts Antifibrotic and Anti-Inflammatory Therapeutic Effects on Perimysial Orbital Fibroblasts in Graves' Orbitopathy.

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-ß1 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-ß1 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.

Disulfiram Exerts Antiadipogenic, Anti-Inflammatory, and Antifibrotic Therapeutic Effects in an In Vitro Model of Graves' Orbitopathy.

Background: Adipogenesis, glycosaminoglycan hyaluronan (HA) production, inflammation, and fibrosis are the main pathogenic mechanisms responsible for Graves' orbitopathy (GO). We hypothesized that disulfiram (DSF), an aldehyde dehydrogenase (ALDH) inhibitor used to treat alcoholism, would have therapeutic effects on orbital fibroblasts (OFs) in GO. This study aimed at determining the therapeutic effects and underlying mechanisms of DSF on these parameters. Methods: Primary cultures of OFs from six GO patients and six control subjects were established. The OFs were allowed to differentiate into adipocytes and treated with various concentrations of DSF. Lipid accumulation within the cells was evaluated by Oil Red O staining. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to measure the expression of key adipogenic transcription factors, ALDH1A1, ALDH2, and mitogen-activated protein kinase (MAPK) signaling proteins. Apoptosis assays and reactive oxygen species levels were evaluated by flow cytometry. HA production was measured by using an enzyme-linked immunosorbent assay (ELISA) kit. The mRNA levels of proinflammatory molecules were measured by using RT-PCR after interleukin (IL)-1ß stimulation with or without DSF. The mRNA expression of markers associated with fibrosis was examined by using RT-PCR after transforming growth factor (TGF)-ß1 stimulation with or without DSF. The wound-healing assay was assessed by phase-contrast microscopy. Results: Under identical adipogenesis conditions, GO OFs effectively differentiated, while normal control (NC) OFs did not. DSF dose dependently suppressed lipid accumulation during adipogenesis in GO OFs. The expression of key adipogenic transcription factors, such as perilipin-1 (PLIN1), PPARγ (PPARG), FABP4, and c/EBPα (CEBPA), was downregulated. Further, DSF inhibited the phosphorylation of ERK by inhibiting ALDH1A1. In addition, DSF attenuated HA production and suppressed inflammatory molecule expression induced by IL-1ß in GO OFs and NC OFs. The antifibrotic effects of DSF on TGF-ß1 were also observed in GO OFs. Conclusions: In the current study, we provide evidence of the inhibitory effect of DSF on GO OFs adipogenesis, HA production, inflammation, and fibrosis in vitro. The results of this study are noteworthy and indicate the potential use of DSF as a therapeutic agent for the treatment of GO.