DOI: 10.5061/dryad.1rn8pk13p
title: Supplementary data from: 2'-O-Galloylhyperin prevents tissue remodeling in thyroid eye disease: prospects as a thyrotropin receptor antagonist

description: 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.
description: # Supplementary Material for 2'-O-Galloylhyperin prevents Tissue
 Remodeling in Thyroid Eye Disease: Prospects as a Thyrotropin Receptor
 Antagonist
 [https://doi.org/10.5061/dryad.1rn8pk13p](https://doi.org/10.5061/dryad.1rn8pk13p) ## Description of the data and file structure ### Files and variables 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* &lt; 0.05 and ***P* &lt; 0.01 versus the DMSO group. ## Code/software * N/A ## Access information Other publicly accessible locations of the data: * N/A Data was derived from the following sources: * N/A