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 to determine the therapeutic effects and underlying mechanisms of DSF on these parameters. Methods Primary cultures of OFs from 6 GO patients and 6 control subjects were established. 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 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 (ROS) levels were evaluated by flow cytometry. HA production was measured using an ELISA kit. The mRNA levels of proinflammatory molecules were measured using real-time PCR after interleukin (IL)-1β stimulation with or without DSF. The mRNA expression of markers associated with fibrosis was examined using real-time PCR after transforming growth factor (TGF)-β1 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 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. Furthermore, DSF inhibited the phosphorylation of ERK by inhibiting ALDH1A1. In addition, DSF attenuated HA production and suppressed inflammatory molecule expression induced by IL-1β in GO OFs and NC OFs. The antifibrotic effects of DSF on TGF-β1 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.