Supplementary data from: 2'-O-Galloylhyperin prevents tissue remodeling in thyroid eye disease: prospects as a thyrotropin receptor antagonist

Context: Thyroid eye disease (TED) is a challenging condition
owing to relentless orbital tissue remodeling, with thyroid-stimulating
hormone receptor (TSHR) in orbital fibroblasts (OFs) serving as a
promising therapeutic target. Objectives: This study seeks to
discover potential TSHR inhibitors among FDA-approved drugs and evaluate
their effects on TED-OFs. Design/Participants: Adipose tissues
were sourced from the patients with or without TED.
Isolated OFs were cultivated in proliferation medium or
stimulated for adipogenic/fibrotic differentiation in 2D/3D
models, treated by 2'-O-GH (0, 5, 20, and 50μM). Main Outcome
Measures: Using structure-based virtual screening (SBVS),
potential TSHR antagonists were identified. Cellular proliferation was
analyzed by EdU incorporation, flow cytometry and spheroid size.
Adipogenesis was determined by Oil Red O staining, Western
blot and immunofluorescence. Fibrosis was assessed using
woundhealing assays, Western blot and immunofluorescence. Cyclic adenosine
monophosphate (cAMP), hyaluronan and cytokine were quantified by ELISA.
Results: Herein, the FDA-approved drug
2'-O-Galloylhyperin (2'-O-GH) dose-dependently decreased cAMP
production and the subsequent CREB
phosphorylation stimulated by a TSHR
stimulating monoclonal autoantibody M22, which was reversed by a
consistently activated mutation of TSHR (L629F). As expected, 2'-O-GH
attenuated lipid accumulation in TED-OFs, along
with down-regulation of key adipogenic markers. And
2'-O-GH ameliorated hyaluronan production during
adipogenesis. Notably, 2'-O-GH
dampened wound closure and fibrotic
differentiation of TED-OF stimualted by TGF-β1. Correspondingly,
proliferation of TED-OFs was halted by 2'-O-GH.
Conclusions: 2'-O-GH promises to prevent the tissue remodeling of TED
by exerting inhibitory effects on proliferation, differentiation and
hyaluronan deposition by inhibiting TSHR activation, implying its
potential therapeutic value for TED.