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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. [ABSTRACT FROM AUTHOR]

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)-1β stimulation with or without DSF. The mRNA expression of markers associated with fibrosis was examined by using RT-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 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-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. [ABSTRACT FROM AUTHOR]

Purpose. To investigate the synergic effect of combination of orbital decompression surgery and methylprednisolone pulse therapy (MPT) and MPT alone on the visual function in patients with bilateral dysthyroid optic neuropathy (DON). Methods. For each involved patient with bilateral DON, only one eye was treated with orbital decompression surgery which was conducted by the same doctor, and each of them received MPT after surgery. If the visual function deteriorated despite treatment, patients would switch to the other treatment. All the patients were followed up for 3 months after surgery. Clinical features of patients including best corrected visual acuity (BCVA), intraocular pressure (IOP), proptosis, upper eyelid retraction, and clinical activity score (CAS) before and after surgery were analyzed, respectively. Visual field and visual evoked potential (VEP) tests were also performed. Paired t-test and Wilcoxon matched-pairs signed ranks sum test were used to analyze the data. Result. A prospective cohort of 23 patients with bilateral DON was enrolled in this cohort study. No patients failed to the therapy or switched to another treatment. The quantitative variables were shown as means and standard deviations (SD). After 3 months of combined treatment of orbital decompression surgery and MPT, BCVA (logMAR) improved, proptosis was reduced and the upper eyelid retraction was relieved in both eyes of patients; however, these improvements were more significant in the operated eyes than in the fellow (nonoperated) eyes. IOP decreased significantly in the operated eyes (P = 0.002), while having no significant change in the nonoperated eyes (P = 0.993). CAS reduced by 0.8 ± 1.37 in the operated eyes and by 0.9 ± 1.28 in the nonoperated eyes (P = 0.011 , P = 0.005 , respectively), but its reduction extent showed no significant difference between the operated and fellow eyes (P = 0.771). Visual field tests showed the mean deviation (MD) of the operated and fellow eyes both increased significantly after 3 months of treatment (P < 0.001 , P = 0.001 , respectively). MD of the operated eyes increased by 8.1 ± 7.72 dB, which was more significant than that of the fellow eyes which increased by 3.4 ± 5.02 dB (P = 0.005). The VEP test showed that, in the operated eyes, the latency of each spatial frequency of P100 was significantly shortened (P < 0.05 , respectively), and the amplitude was significantly improved (P < 0.05 , respectively); however, there was no significant change in VEP parameters of the fellow eyes (P > 0.05 , respectively). The latency in the operated eyes improved by 28.1 ± 29.93 ms in 60′ P100, by 40.2 ± 32.87 ms in 30′ P100, and by 20.7 ± 25.87 ms in 15′ P100 respectively, which was more excellent in the degree of the improvement than that in the fellow eyes (P = 0.002 , P = 0.001 , P = 0.005 , respectively). Conclusion. A combination of orbital decompression and MPT can significantly improve visual function in patients with DON, reduce intraocular pressure, and relieve clinical symptoms such as upper eyelid retraction and proptosis, while MPT alone has a limited effect. For DON patients, orbital decompression should be performed promptly to improve the visual function. [ABSTRACT FROM AUTHOR]