Supplementary data from: 2'-O-Galloylhyperin prevents tissue remodeling in thyroid eye disease: prospects as a thyrotropin receptor antagonist
Abstract
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.
https://doi.org/10.5061/dryad.1rn8pk13p
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This is supplemental materials for the published article entitled “2’-O-Galloylhyperin prevents Tissue Remodeling in Thyroid Eye Disease: Prospects as a Thyrotropin Receptor Antagonist”.
Supplemental Figure 1. Expression of TSHR in orbital adipose. (images on Zenodo)
(A) Representative IF images of TSHR expression in orbital adipose tissues from the patients with or without TED. Similar images were obtained from three independent OF samples. Scale bar: 100μm.
(B) Immunoblots with TSHR in OFs from patients with or without TED (n = 3). GAPDH served as the control.
Supplemental Figure 2. The extra effects of 2’-O-GH on inflammation and oxidative stress
(A) ELISA assays for IL-6 secretion in TED-OFs added with DM, TGF-β1 or IL-1β, and treated with 2’-O-GH at the indicated concentrations for 48 hours (n = 3).
(B) ELISA assays for IL-8 secretion in TED-OFs added with DM, TGF-β1 or IL-1β, and treated with 2’-O-GH at the indicated concentrations for 48 hours (n = 3).
(C) Representative fluorescent images of fluorescence of 2’-7’-dichlorofluorescein (DCF) in TED-OFs administered with TGF-β1, treated with 2’-O-GH at the indicated concentrations. Similar images were obtained from three independent OF samples. Scale bar: 200μm.
(D) Quantification of the fluorescent intensity in (C), as measured by the integrated density (n=3).
For (A), (B) and (D), values are means ± SEM. *P < 0.05 and **P < 0.01 versus the DMSO group.
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