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Eye Movements, Strabismus, Amblyopia and Neuro-ophthalmology  |   June 2023
The Potential of Artemisinins as Novel Treatment for Thyroid Eye Disease by Inhibiting Adipogenesis in Orbital Fibroblasts
Author Affiliations & Notes
  • Yan Guo
    Department of Endocrinology and Diabetes Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
  • Yanglei Cheng
    Department of Endocrinology and Diabetes Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
  • Hai Li
    Department of Endocrinology and Diabetes Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
  • Hongyu Guan
    Department of Endocrinology and Diabetes Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
  • Haipeng Xiao
    Department of Endocrinology and Diabetes Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
  • Yanbing Li
    Department of Endocrinology and Diabetes Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
  • Correspondence: Yanbing Li, 58 Zhongshan Road II, Guangzhou, Guangdong 510080, China; [email protected]
  • Footnotes
     YG, YC, and HL contributed equally to this work.
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 28. doi:https://doi.org/10.1167/iovs.64.7.28
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      Yan Guo, Yanglei Cheng, Hai Li, Hongyu Guan, Haipeng Xiao, Yanbing Li; The Potential of Artemisinins as Novel Treatment for Thyroid Eye Disease by Inhibiting Adipogenesis in Orbital Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2023;64(7):28. https://doi.org/10.1167/iovs.64.7.28.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Thyroid eye disease (TED) causes cosmetic defect and even threatens eyesight due to tissue remodeling in which orbital fibroblast (OF) plays a central role mainly by differentiating into adipocytes. Repurposing old drugs to novel applications is of particular interest. Here, we aimed to evaluate the effects of the antimalarials artemisinin (ARS) and the derivatives on the OFs isolated from patients with TED and their counterparts.

Methods: OFs isolated from patients with TED or their counterparts were cultured and passaged in proliferation medium (PM) and stimulated by differentiation medium (DM) for adipogenesis. OFs were treated with or without ARS, dihydroartemisinin (DHA), and artesunate (ART) at different concentrations, before being examined in vitro. CCK-8 were used to assess cellular viability. Cell proliferation was determined by EdU incorporation and flow cytometry. Lipid accumulation within the cells was evaluated by Oil Red O staining. Hyaluronan production was determined by ELISA. RNAseq, qPCR, and Western blot analysis were performed to illustrate the underlying mechanisms.

Results: ARSs dose-dependently interfered with lipid accumulation of TED-OFs, rather than non-TED-OFs. Meanwhile, the expression of key adipogenic markers, such as PLIN1, PPARG, FABP4, and CEBPA, was suppressed. During adipogenesis as being cultivated in DM, instead of PM, ARSs also inhibited cell cycle, hyaluronan production and the expression of hyaluronan synthase 2 (HAS2) in a concentration-dependent manner. Mechanically, the favorable effects were potentially mediated by the repression of IGF1R-PI3K-AKT signaling by dampening IGF1R expression.

Conclusions: Collectedly, our data evidenced that the conventional antimalarials ARSs were potentially therapeutic for TED.

Thyroid eye disease (TED) is the most common extrathyroidal symptom of Graves’ disease.1 Mainly manifested as proptosis and strabismus et al, TED negatively impacts patients’ appearance, psychosocial wellbeing, social functions, and even exacerbates visual impairment in 3% to 5% of cases, which imposes a crushing burden on the individual and society.2,3 Unfortunately, the pathogenesis of TED is unclear, such that therapeutic options for TED especially proptosis are currently limited.1 For active TED, the effective rate of the mainstream therapy intravenous glucocorticoids (GCs) is approximately 60%, along with side effects, such as hyperglycemia and osteoporosis. Mild inactive TED benefits little from medical treatments, which tends to be remedied with surgery due to persistent exophthalmos and cosmetic concerns.4 It remains to be a critical unmet clinical requirement to discover more effective and economical treatments for TED exophthalmos. 
Orbital fibroblast (OF) has been regarded as the main effector cell involved in the intraorbital tissue remodeling.4 OFs undergo robust proliferation, excessive adipogenic differentiation (adipogenesis), and hyaluronan (HA) production in the pathological microenvironment of TED. Valyasevi et al. found that the peroxisome proliferators-activated receptor gamma (PPARG) agonist rosiglitazone exacerbates the exophthalmos symptoms of TED by promoting adipogenesis.5,6 Nonetheless, anti-adipogenic treatment has yet to be clinically applied to TED therapy. Our previous study showed that the antimalarials chloroquine (CQ) and hydroxychloroquine (HCQ) exert anti-adipogenic and antiproliferative functions, and inhibit HA secretion in OFs from patients with TED.7 Given the high costs in drug research and development, the repurposing of existing medications to extend their clinical indications is of particular interest.8 
Artemisinin (ARS) is commonly used as an antimalaria drug worldwide after CQ.9 Dihydroartemisinin (DHA) and artesunate (ART) are two major water-soluble semisynthetic derivatives of ARS, which have been well accepted as the first-line antimalarials with little severe side effects.10,11 Apart from their antimalarial mode of action, ARS and the derivatives (ARSs) have played important roles in various pathological process, such as tumorigenesis,12 respiratory diseases, such as chronic obstructive pulmonary disease (COPD),13 and autoimmune diseases, such as rheumatoid arthritis (RA).14 ARSs have also been recently regarded as a promising treatment for coronavirus disease 2019 (COVID-19).15,16 Moreover, it has been revealed by recent studies that DHA and ART showed antagonistic action on adipogenesis of 3T3-L1 pre-adipocytes.17,18 Herein, we attempt to determine whether ARSs elicit novel effects on TED and the underlying mechanisms using OFs from patients with TED and non-TED patients, which is a widely accepted in vitro model for TED studies. 
Materials and Methods
Adipose Tissue Collection and Ethics Statements
Orbital adipose tissue samples were collected from 10 patients diagnosed with TED, and from 9 non-TED participants during surgery at Zhongshan Ophthalmic Center, Sun Yat-Sen University, listed in Tables 1 and 2, respectively. All tissue specimens were obtained in accordance with the principles set out in the Declaration of Helsinki, with the approval of the Institutional Review Board of Zhongshan Ophthalmic Center and the institutional human research ethics committee in the First Affiliated Hospital of Sun Yat-Sen University. Written informed consent was obtained from each patient. 
Table 1.
 
Clinical Characteristics of Patients With TED in the Study
Table 1.
 
Clinical Characteristics of Patients With TED in the Study
Table 2.
 
Clinical Characteristics of Patients Without TED in the Study
Table 2.
 
Clinical Characteristics of Patients Without TED in the Study
OF Isolation and Culture
OFs were isolated as before.7 OFs were cultivated with proliferation medium (PM), which is Dulbecco's modified Eagle's medium nutrient mixture F-12 (Ham; DMEM-F12) supplemented with 10% (vol/vol) fetal bovine serum (FBS; Gibco Laboratories, USA), 100 IU/mL penicillin and 100 mg/mL streptomycin, at 37°C in a 5% CO2 humidified incubator. Each experiment was repeated using OFs from at least three independent specimens. OFs from both sexes were used without preference. 
Flow Cytometry
OFs were incubated with the appropriate antibody in the dark at 4°C for 30 minutes and analyzed by flow cytometry using Influx flow cytometers (BD Biosciences, USA). The data were analyzed using CytExpert software. The antibodies for flow cytometry were listed in Supplementary Table S1
Adipogenic Differentiation
To induce adipogenic differentiation, PM was changed to the commercial adipogenic differentiation media (DM; #S-09-007, SALIAI, China) after cells reached confluence for 48 hours. The medium was replaced with fresh DM every 2 to 3 days according to the instruction manual. ARS/DHA/ART (#A2118, #D3793, and #A2191; TCI, China) were administrated 24 hours before DM cultivation and kept for 10 days. 
Oil Red O Staining
The differentiated OFs were fixed and stained with Oil Red O working solution (SALIAI, China) for 30 minutes. The images were photographed using a microscope (Olympus IX71, Tokyo, Japan). To quantify lipid accumulation, isopropanol was added to the stained OFs. The optical density (OD) of each well was measured at 450 nm using a Bioteck Gene5 microplate reader (Bioteck, USA). Details were shown as previously reported.7 
Cell Counting Kit-8 Assays
Cellular viability was assessed using a CCK-8 assay kit (KeyGEN BioTECH, China). The OD values (450 nm) were measured by a Bioteck Gene5 microplate reader (Bioteck, USA). Details were shown as previously reported.7 
Cell Proliferation Assays
The 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay and cell cycle analysis were performed as previously reported.19 
Western Blot Analysis
Western blot analysis was performed as described previously.19 The antibodies are listed in Supplementary Table S1. The intensity of each band was calculated with ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to that of GAPDH. 
Measurement of HA Production
HA was measured with an enzyme-linked immunosorbent assay (ELISA) kit (Echelon Bioscience Inc., USA) according to the manufacturer's instructions. Details were described as previously reported.7 
RNA Extraction, Reverse Transcription-Polymerase Chain Reaction and Quantitative Real-Time PCR
Total RNA was extracted with TRIzol Reagent (Life Technologies, USA). One-step RT-PCR reaction was performed using an RT-PCR kit (Promega, USA), and qPCR was conducted using LightCycle480 II (Roche, USA), as described in a previous study.19 The primers used for qPCR are listed in Supplementary Table S2
RNA Sequencing and Functional Enrichment Analysis
Total RNA extracted from TED-OFs in PM+DMSO (PM), DM+DMSO (DM), and DM+ARS (ARS) was evaluated using Agilent 2100 BioAnalyzer (Agilent Technologies) and Qubit Fluorometer (Invitrogen). Total RNA samples with RNA integrity number (RIN) > 7.0 and 28S:18S ratio > 1.8 were used in the subsequent generation and sequencing of RNA-seq libraries by CapitalBio Technology. Enrichment analysis was performed using KOBAS3.0 (http://kobas.cbi.pku.edu.cn/genelist/). Gene sets with adjusted P < 0.05 were defined as statistically significant. 
Statistical Analysis
Each experiment involved the OFs isolated from three to six patients. The data are presented as the mean ± SEM and were analyzed by applying independent-samples T tests, nonparametric tests (Mann-Whitney U Tests) or one-way analysis of variance (ANOVA; LSD) using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). Statistical significance required a P value < 0.05. 
Results
Isolation and Identification of OFs
Primary OFs were isolated from orbital connective tissues separated from patients with TED and non-TED patients, respectively (Fig. 1A). The isolated OFs which belong to the MSC family, were identified by the surface markers CD44, CD73, CD90, and CD105, whereas those of other cell types, such as CD45, human leukocyte antigen DR (HLA-DR), CD19, and CD11b, were negatively expressed (Fig. 1B). As shown in Figure 1C, the OFs cultured in vitro could stably maintain the morphology and viability after at least five time passages. 
Figure 1.
 
Isolation and identification of OFs. (A) OFs were obtained from stromal vascular fraction (SVF) of orbital adipose tissues after collagenase digestion. (B) The surface marker expressions of TED-OFs (CD44, CD73, CD90, and CD105), hematopoietic cells (CD45), T lymphocytes (HLA-DR), B lymphocytes (CD19), and macrophages (CD11b) were analyzed by flow cytometry. (C) Relative microscopic images of TED-OFs of the indicated passages.
Figure 1.
 
Isolation and identification of OFs. (A) OFs were obtained from stromal vascular fraction (SVF) of orbital adipose tissues after collagenase digestion. (B) The surface marker expressions of TED-OFs (CD44, CD73, CD90, and CD105), hematopoietic cells (CD45), T lymphocytes (HLA-DR), B lymphocytes (CD19), and macrophages (CD11b) were analyzed by flow cytometry. (C) Relative microscopic images of TED-OFs of the indicated passages.
Nontoxic Concentrations of ARS and the Derivatives were Confirmed
We involved ARS, the derivatives ART with considerable water-solubility, and DHA which is the metabolite of the former two in vivo (Fig. 2A). To explore the noncytotoxic concentrations of ARS and the derivatives in OFs, CCK-8 assays were conducted. As seen in Figures 2B, 2C, and 2D, ARS showed low cytotoxicity to OFs at concentrations ≤ 200 µM for 24, 48, and 72 hours, whereas DHA ≤ 20 µM and ART ≤ 10 µM were harmless for OFs. In addition, ARS at 200 µM, DHA at 20 µM, and ART at 10 µM were innoxious to OFs from non-TED patients for 24, 48, and 72 hours, shown in Figure 2E. As a result, different dosages of ARS (10, 50, and 200 µM), DHA (1, 5, and 20 µM), and ART (0.5, 2, and 10 µM), were chosen for the subsequent experiments. 
Figure 2.
 
Cellular cytotoxicity of ARS and the derivatives at different concentrations was tested by CCK-8 assays. (A) Chemical structures of three artemisinin-based drugs (ARSs). (B–D) CCK-8 assays were conducted to analyze cell cytotoxicity of TED-OFs treated with different concentration gradients of (B) ARS (0, 10, 25, 50, 100, 200, 500, and 1000 µM), (C) DHA (0, 0.5, 1, 5, 10, 20, 50, and 100 µM), and (D) ART (0, 0.5, 1, 5, 10, 20, 50, and 100 µM) for 24, 48, and 72 hours, respectively (n = 6 per group). (E) OFs from non-TED patients (non-TED-OFs) were treated with of ARS (200 µM), DHA (20 µM), and ART (10 µM) for 24, 48, and 72 hours, respectively (n = 6 per group). Relative CCK-8 intensity is presented as the percentage normalized to the average OD value of the DMSO-treated wells. The bar graph data are shown as the mean ± SEM from 6 independent TED-OF samples. *P < 0.05, **P < 0.01, ***P < 0.001, nsP ≥ 0.05 versus DMSO group.
Figure 2.
 
Cellular cytotoxicity of ARS and the derivatives at different concentrations was tested by CCK-8 assays. (A) Chemical structures of three artemisinin-based drugs (ARSs). (B–D) CCK-8 assays were conducted to analyze cell cytotoxicity of TED-OFs treated with different concentration gradients of (B) ARS (0, 10, 25, 50, 100, 200, 500, and 1000 µM), (C) DHA (0, 0.5, 1, 5, 10, 20, 50, and 100 µM), and (D) ART (0, 0.5, 1, 5, 10, 20, 50, and 100 µM) for 24, 48, and 72 hours, respectively (n = 6 per group). (E) OFs from non-TED patients (non-TED-OFs) were treated with of ARS (200 µM), DHA (20 µM), and ART (10 µM) for 24, 48, and 72 hours, respectively (n = 6 per group). Relative CCK-8 intensity is presented as the percentage normalized to the average OD value of the DMSO-treated wells. The bar graph data are shown as the mean ± SEM from 6 independent TED-OF samples. *P < 0.05, **P < 0.01, ***P < 0.001, nsP ≥ 0.05 versus DMSO group.
Adipogenesis of TED-OFs was Perturbed by ARS and the Derivatives
To determine the anti-adipogenic effects of ARS and the derivatives on the cultivated OFs from patients with TED and non-TED patients, OFs were stimulated by DM for 10 days treated with ARS, DHA, and ART at the indicated concentrations. As illustrated in Figure 3A, typical morphological changes, such as cell rounding and accumulation of large lipid droplets, as well as intensive staining of Oil Red O (ORO) were observed in the TED-OFs after 10-day induction, whereas those from non-TED patients tended to display with spindle shape. As expected, TED-OFs treated by ARS and the derivatives showed different levels of differentiation abnormality depending on the dosages. As shown in Figure 3B, ORO staining calculated as the OD value, was significantly reduced by ARS in TED-OFs in a concentration-dependent way (ARS versus DMSO: 10 µM [P = 0.0001], 50 µM [P = 0.0001], 200 µM [P = 0.0001]), whereas DHA and ART exerted the analogous but weaker anti-adipogenic effect at the concentration of 20 µM (P = 0.0245) and 10 µM (P = 0.0047), respectively (Fig. 3B). However, no obvious difference was detected in non-TED-OFs among groups treated with or without ARS (200 µM, P = 0.9155), ART (10 µM, P = 0.6711), and DHA (20 µM, P = 0.9592; see Figs. 3A, 3C). Furthermore, we performed Western blot analysis to detect pro-adipogenic and mature adipocyte markers. In line with the results in ORO staining, ARS and the derivatives markedly decreased the expressions of the adipogenetic markers PLIN1, PPARG, CCAAT enhancer-binding protein alpha (CEBPA), and FABP4, in a dose-dependent way, as shown in Figures 3D–G. In order to exclude the possibility that ARSs attenuated adipogenesis of OFs via cytotoxic effect, a CCK-8 assay was conducted on TED- and non-TED-OFs under the same conditions (Supplementary Fig. S1A). 
Figure 3.
 
Effects of ARS and the derivatives on adipogenesis of OFs in vitro. Forty-eight hours after growth arrest, the confluent TED-OFs were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with differentiation media (DM) containing ARSs at the indicated concentrations for 10 days. (A) Representative microscopic images of Oil Red O (ORO) staining in adipogenic OFs from patients with TED and non-TED patients. Scale bars = 400 µm. Similar images were obtained from six independent TED-OF samples. (B) Quantification of the ORO staining for TED-OFs in (A), as measured by the OD values (450 nm) after stained cells were solubilized (n = 6 per group). (C) Quantification of the ORO staining for non-TED-OFs in (A), as measured by the OD values (450 nm) after stained cells were solubilized (n = 6 per group). (D) Representative immunoblots with the adipogenesis markers in TED-OFs was assessed by Western blot analysis. GAPDH served as the control. Similar images were obtained from four independent TED-OF samples. (E–G) The protein levels were quantified, normalized to the level of GAPDH for each sample and analyzed (n = 4 per group). For (B), (C), and (E–G), the results are shown as the mean ± SEM. *P < 0.05, **P < 0.01. ***P < 0.001, nsP ≥ 0.05 versus the DMSO group.
Figure 3.
 
Effects of ARS and the derivatives on adipogenesis of OFs in vitro. Forty-eight hours after growth arrest, the confluent TED-OFs were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with differentiation media (DM) containing ARSs at the indicated concentrations for 10 days. (A) Representative microscopic images of Oil Red O (ORO) staining in adipogenic OFs from patients with TED and non-TED patients. Scale bars = 400 µm. Similar images were obtained from six independent TED-OF samples. (B) Quantification of the ORO staining for TED-OFs in (A), as measured by the OD values (450 nm) after stained cells were solubilized (n = 6 per group). (C) Quantification of the ORO staining for non-TED-OFs in (A), as measured by the OD values (450 nm) after stained cells were solubilized (n = 6 per group). (D) Representative immunoblots with the adipogenesis markers in TED-OFs was assessed by Western blot analysis. GAPDH served as the control. Similar images were obtained from four independent TED-OF samples. (E–G) The protein levels were quantified, normalized to the level of GAPDH for each sample and analyzed (n = 4 per group). For (B), (C), and (E–G), the results are shown as the mean ± SEM. *P < 0.05, **P < 0.01. ***P < 0.001, nsP ≥ 0.05 versus the DMSO group.
ARS and the Derivatives Played Anti-Proliferative Role on Mitotic Clonal Expansion of TED-OFs
We have demonstrated that the confluent TED-OFs also re-entered the cell cycle at the early stage of adipogenesis, termed as mitotic clonal expansion (MCE), a physiological process prerequisite for adipogenesis of 3T3-L1, the preadipocyte cell line.7,20,21 Given the anti-adipogenic effect of ARS and the derivatives, we were prompted to analyze their impacts on MCE process. Intriguingly, ARS, DHA, and ART remarkably halted the MCE progression via arresting cell cycle at the G0/G1 phase in a concentration-dependent manner (ARS [200 µM] versus DMSO: P = 0.0284; ART [10 µM] versus DMSO: P = 0.0042; DHA [20 µM] versus DMSO: P = 0.0011), as demonstrated by flow cytometry analyses (Figs. 4A, 4B). Consistently, as shown in Figures 4C and 4D, ARS, DHA, and ART dose-dependently prevented TED-OFs instead of non-TED-OFs from regaining DNA synthesis ability after growth arrest, reflected by EdU assays (ARS [200 µM] versus DMSO: P = 0.002; ART [10 µM] versus DMSO: P = 0.0001; DHA [20 µM] versus DMSO: P = 0.0002). However, no significant difference in the EdU-positive ratio of TED-OFs treated by ARS/DHA/ART in PM without adipogenic stimuli, as shown in Supplementary Figures S2A and S2B (ARS [200 µM] versus DMSO: P = 0.9474; ART [10 µM] versus DMSO: P = 0.4141; DHA [20 µM] versus DMSO: P = 0.6778). It was suggested that ARS and the derivatives exerted cytostatic effect specifically at the early stage of adipogenesis, potentially contributed to the antiadipogenic phenotype. As shown in Supplementary Figure S1B, ARSs added in DM for 20 hours did not alter cell viability in either TED- or non-TED-OFs. 
Figure 4.
 
Effects of ARS and the derivatives on the cellular proliferation of OFs in DM. Forty-eight hours after growth arrest, the confluent OFs (from patients with TED or non-TED patients) were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with DM for 20 hours. (A) Cartogram about cell cycle distribution of each TED-OFs group treated with or without ARS, DHA, and ART at the indicated concentrations in DM (n = 5 per group). (B) Representative experimental diagrams of the cell cycle distribution of each TED-OFs group treated with DMSO, ARS (200 µM), DHA (20 µM), and ART (10 µM) in DM, as determined by flow cytometry. (C) Representative images of the EdU incorporation assay results in each group of TED- and non-TED-OFs treated with DMSO, ARS (200 µM), DHA (20 µM), and ART (10 µM) in DM, scale bars = 100 µm. Red: EdU, Blue: DAPI. (D) Quantification of the EdU incorporation assays for (C), n = 5 per group. For (A) and (D), the data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, nsP ≥ 0.05 versus the DMSO group.
Figure 4.
 
Effects of ARS and the derivatives on the cellular proliferation of OFs in DM. Forty-eight hours after growth arrest, the confluent OFs (from patients with TED or non-TED patients) were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with DM for 20 hours. (A) Cartogram about cell cycle distribution of each TED-OFs group treated with or without ARS, DHA, and ART at the indicated concentrations in DM (n = 5 per group). (B) Representative experimental diagrams of the cell cycle distribution of each TED-OFs group treated with DMSO, ARS (200 µM), DHA (20 µM), and ART (10 µM) in DM, as determined by flow cytometry. (C) Representative images of the EdU incorporation assay results in each group of TED- and non-TED-OFs treated with DMSO, ARS (200 µM), DHA (20 µM), and ART (10 µM) in DM, scale bars = 100 µm. Red: EdU, Blue: DAPI. (D) Quantification of the EdU incorporation assays for (C), n = 5 per group. For (A) and (D), the data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, nsP ≥ 0.05 versus the DMSO group.
ARS and the Derivatives Suppressed HA Production During Adipogenesis in TED-OFs
It was reported that HA is synthesized and secreted by OFs along with adipogenesis.22 Hence, we further examined whether HA production was attenuated by ARS and the derivatives. Expectedly, HA production in TED-OFs with DM manipulation was lowered by ARS/DHA/ART at the indicated concentrations (ARS [200 µM] versus DMSO: P = 0.0297; ART [10 µM] versus DMSO: P = 0.0074; DHA [20 µM] versus DMSO: P = 0.0160), which was not the case in non-TED-OFs (Fig. 5A). According to the studies reported before, DM-stimulated HA synthesis is mediated mainly by hyaluronan synthase 2 (HAS2) upregulation.7,23 As presented in Figures 5B–F, HAS2 expression was also decreased by ARS and the derivatives (ARS [200 µM] versus DMSO: P = 0.0139; ART [10 µM] versus DMSO: P = 0.0332; DHA [20 µM] versus DMSO: P = 0.0458). In addition, ART lowered HAS3 levels (ART [0.5 µM] versus DMSO: P = 0.0100; ART [2 µM] versus DMSO: P = 0.0096; ART [10 µM] versus DMSO: P = 0.0087) and hyalase 2 (HYAL2) expression (ART [2 µM] versus DMSO: P = 0.0241; ART [10 µM] versus DMSO: P = 0.0245) in a dose-dependent way. Although there was no significant difference in HAS1 and HYAL1 expression among different groups. In addition, ARSs displayed no obvious cytotoxicity in TED- or non-TED-OFs under the conditions used in the HA generation experiments (see Supplementary Fig. S1C). 
Figure 5.
 
Effects of ARS and the derivatives on the HA production of OFs in DM. Forty-eight hours after growth arrest, the confluent OFs (from patients with TED or non-TED patients) were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with DM for 48 hours, and then the supernatants were collected for ELISA analysis. (A) HA secretion in each TED (n = 5) and non-TED (n = 4) group were determined by ELISA. (B–F) The mRNA levels of HAS1 (B), HAS2 (C), HAS3 (D), HYAL1 (E), and HYAL2 (F) relative to those of GAPDH (normalized to the average values of the DMSO group), detected by qPCR in TED-OFs treated with or without ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 96 hours in DM. (B: n = 5; C–F: n = 3). The data are shown as the mean ± SEM, *P < 0.05, **P < 0.01, nsP ≥ 0.05 versus the DMSO group.
Figure 5.
 
Effects of ARS and the derivatives on the HA production of OFs in DM. Forty-eight hours after growth arrest, the confluent OFs (from patients with TED or non-TED patients) were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with DM for 48 hours, and then the supernatants were collected for ELISA analysis. (A) HA secretion in each TED (n = 5) and non-TED (n = 4) group were determined by ELISA. (B–F) The mRNA levels of HAS1 (B), HAS2 (C), HAS3 (D), HYAL1 (E), and HYAL2 (F) relative to those of GAPDH (normalized to the average values of the DMSO group), detected by qPCR in TED-OFs treated with or without ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 96 hours in DM. (B: n = 5; C–F: n = 3). The data are shown as the mean ± SEM, *P < 0.05, **P < 0.01, nsP ≥ 0.05 versus the DMSO group.
Interleukin-1β (IL-1β) is another intensive secretagogue of HA, shown in Supplementary Figure S2C. To our surprise, neither DHA nor ART exerted significant effect on HA production with or without stimulation of IL-1β. Whereas, ARS at the concentration of 200 µM, showed repressive effect with statistical significance, on HA release with or without stimulation of IL-1β (see Supplementary Fig. S2C). 
ARS and the Derivatives Blunted IGF1-AKT-FOXO1 Cascade Signaling
To identify the underlying mechanisms mediating the anti-adipogenic role played by ARS in TED-OFs, we used bulk RNA sequencing (RNAseq). At the beginning, we focused on identifying key signaling pathways that are differentially expressed during adipogenic differentiation of TED-OFs in vitro. There were 1398 upregulated genes and 1709 downregulated genes in the TED-OFs induced by DM compared to those in PM (Supplementary Fig. S3A). As depicted in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, multiple regulatory signaling of differentially expressed genes (DEGs), such as proliferation (PI3K/AKT and cell cycle), and energy metabolism (AMPK and FOXO) were enriched in the DM group, apart from the specific adipogenic signaling, such as PPAR signaling and unsaturated fatty acid biosynthesis (see Supplementary Fig. S3B). Using Protein Analysis Through Evolutionary Relationships (PANTHER) enrichment analysis, insulin/IGF1-protein kinase B (PKB or AKT) were observed in the DM group (see Supplementary Fig. S3C). Compared with those in the DM treated group without ARS, a total of 86 upregulated DEGs and 74 downregulated DEGs were detected in TED-OFs in the DM treated group with ARS (Fig. 6A). As analyzed by KEGG of DEGs, PPAR signaling and fatty acid metabolism were affected in the DM+ARS group (Fig. 6B). Using PANTHER analysis, insulin/IGF1 cascade signaling were highlighted (Fig. 6C). Furthermore, upregulated DEGs in the DM versus the PM data set and downregulated ones in the ARS versus the DM data set were compared via online Venn diagram tools (Venny version 2.1). As illustrated in Figure 6D and Supplementary Table S3, we identified 50 oppositely regulated DEGs. Similarly, we compared downregulated DEGs in the DM versus the PM data set and those upregulated by ARS to identify 19 oppositely regulated DEGs (see Fig. 6D, Supplementary Table S4). Given that the remarkable impacts of ARS on insulin/IGF1 signaling and fatty acid metabolism which are involved in adipogenesis and lipogenesis, we focused on IGF1R and stearoyl-CoA desaturase (SCD). As shown in Figure 6E, the qPCR was performed to confirm that mRNA levels of IGF1R were suppressed by ARS, DHA, and ART in a concentration-dependent manner (ARS versus DMSO: 10 µM [P = 0.0808], 50 µM [P = 0.0269], 200 µM [P = 0.0086]; ART versus DMSO: 0.5 µM [P = 0.1578], 2 µM [P = 0.0379], 10 µM [P = 0.0235]; DHA versus DMSO: 1 µM [P = 0.0527], 5 µM [P = 0.0277], and 20 µM [P = 0.0152]). Consistently, mRNA levels of SCD were suppressed by ARS, DHA, and ART concentration dependently. We perform Western blot analysis to analyze whether ARSs interfere with the signaling of PI3K-AKT-FOXO1 pathway, which is downstream of IGF1R. As expected, phosphorylation of AKT and FOXO1 was inhibited by ARS, DHA, and ART in TED-OFs instead of non-TED-OFs, confirmed by Western blot analysis (Fig. 6F). It was suggested that the inhibitory effect of ARSs on TED-OFs was potentially mediated by the IGF1R-PI3K-AKT-FOXO1 pathway by decreasing IGF1R expression. 
Figure 6.
 
The mechanisms underlying in the effects of ARSs on the adipogenic TED-OFs. For (A–D) 48 hours after growth arrest, the confluent TED-OFs (n = 3) were pretreated by DMSO or ARS (200 µM) for 24 hours, before being added with DM for 96 hours. (A) Heat map of the DEGs in ARS versus DMSO. (B) KEGG pathway enrichment analysis for DEGs in ARS versus DMSO. (C) PANTHER pathway enrichment analysis for DEGs in ARS versus DMSO. (D) Venn diagram comparing the oppositely regulated DEGs in DM versus PM and ARS versus DMSO. (E) The mRNA levels of IGF1R (n = 3) and SCD (n = 3) relative to those of GAPDH (normalized to the average values of the DMSO group), detected by qPCR in TED-OFs treated with or without ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 96 hours in DM. Values are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the DMSO group. (F) Representative immunoblots with AKT and FOXO1 (phosphorylated and total) in TED- and non-TED-OFs was assessed by Western blot analysis. GAPDH served as the control. Similar images were obtained from three independent OF samples.
Figure 6.
 
The mechanisms underlying in the effects of ARSs on the adipogenic TED-OFs. For (A–D) 48 hours after growth arrest, the confluent TED-OFs (n = 3) were pretreated by DMSO or ARS (200 µM) for 24 hours, before being added with DM for 96 hours. (A) Heat map of the DEGs in ARS versus DMSO. (B) KEGG pathway enrichment analysis for DEGs in ARS versus DMSO. (C) PANTHER pathway enrichment analysis for DEGs in ARS versus DMSO. (D) Venn diagram comparing the oppositely regulated DEGs in DM versus PM and ARS versus DMSO. (E) The mRNA levels of IGF1R (n = 3) and SCD (n = 3) relative to those of GAPDH (normalized to the average values of the DMSO group), detected by qPCR in TED-OFs treated with or without ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 96 hours in DM. Values are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the DMSO group. (F) Representative immunoblots with AKT and FOXO1 (phosphorylated and total) in TED- and non-TED-OFs was assessed by Western blot analysis. GAPDH served as the control. Similar images were obtained from three independent OF samples.
Discussion
It remains a challenge to resolve exophthalmos and disfigurement because there is limited available therapeutic options. Herein, we demonstrated that ARS and the derivatives halted adipogenesis, MCE, and HA production in TED-OFs, apart from their conventional role as antimalarials. IGF1 signaling pathway and fatty acid metabolism are potentially involved in the favorable effects. 
Abundant evidences have supported the concept that ARSs could serve as versatile traditional drugs beyond the antimalarial effect, via the antineoplastic, immunoregulating, and anti-infectious functions.12,14,15 Albeit, it was in the in vitro model that ARSs were found to play an antiadipogenic role, it has offered a therapeutic implication in TED therapy. As reported by Wang et al., ART exerted a protective effect on an endotoxin-induced uveitis (EIU) rat model,24 implying that it was concentrated enough in orbit to exert therapeutic effects in vivo. Note of worthy, sensibility to ARSs is different among cell types, and is highly dependent on the expression of transferrin receptor (TFR).25 As shown in the transcriptomic profiling of TED-OFs (DM versus PM) here, TFR is highly expressed in adipogenic TED-OFs in DM, whereas barely detected in those undifferentiated ones (see Supplementary Fig. S3D). This explains to some degree why the favorable effects of ARSs were observed in TED-OFs upon DM instead of PM cultivation in our study. Therefore, ARSs have the potential to have relative specificity to the orbital adipose, which makes it clinically practicable and safe to repurpose ARSs in TED therapy. 
Expression of IGF1R is increased in the orbital tissues of patients with TED, and the specific auto-antibodies are blamed for the excessive activation of OFs.26 The inductive effects of IGF1 on HA production, cellular proliferation, and adipogenesis are also indicated.27 Teprotumumab has been approved by the US Food and Drug Administration (FDA) for TED therapy as a monoclonal antibody targeting IGF1R. Numerous studies have shown that teprotumumab holds promise to attenuate proptosis and clinical activity score (CAS) in moderate-severe active TED by repressing HA synthesis and adipogenesis of OFs.28,29 To the best of our knowledge, adipogenesis includes MCE at the early stage, adipogenic markers upregulation at the medium stage, and the late-stage lipid accumulation mediated by lipogenesis-related genes,30 in which the IGF1-PI3K-AKT-FOXO1 signaling pathway plays a vital role in proliferation and fatty acid metabolism.31 Activation of PI3K-AKT increases the transcription factor sterol regulatory element-binding protein (SREBP), which is up-stream of the important de novo lipogenesis (DNL), genes fatty acid synthase (FASN), and SCD.31 Based on the bioinformatic analysis, IGF1 signaling pathway, was remarkably affected by ARS. Concurrent with what was reported before,32,33 IGF1R and SCD were upregulated in TED-OFs upon DM. We found that they were synergistically lowered in the ARS-treated TED-OFs during adipogenesis. Besides, we also observed that ARS and the derivatives exerted inhibitory effect on HA production of TED-OFs by decreasing HAS2 expression during adipogenesis. Notably, lower HA and HAS2 levels could also dampen adipogenesis in 3T3–L1.34 Collectedly, it was strongly implied that inhibition of IGF1-PI3K-AKT signaling potentially mediated the repressive effects of the antimalarials on MCE, adipogenesis, and HA production in TED-OFs. 
There are some limitations in this study. We have only focused on in vitro studies using OFs from patients with TED and non-TED patients, regardless of the potential role of ARS/DHA/ART in the immunologically active cells and the microenvironment. Given that they were reported to elicit immunomodulatory effects,35 the investigation about their therapeutic effects on TED animal models and humans is indispensable before repurposing ARSs as a therapeutic option for TED. Because ARSs may represent side effects such as nausea, vomiting, and dizziness, it is of great importance to discuss whether the involved concentrations could be achievable and safe by ARSs therapy in further study.36 Despite this, our data has proposed a tempting idea that the conventional drugs could be used to decrease IGF1R expression in TED-OFs. 
In conclusion, ARS and the derivatives exert antiadipogenic and cytostatic effects on TED-OFs, and meanwhile reduce the HA production, which holds promise for repurposing the antimalarials to tackle exophthalmos in TED therapy. 
Acknowledgments
The authors thank Huangsheng Yang (Sun Yat-Sen University, China, Guangzhou) for his help with the collection of tissue specimens and the relative experiments of OFs. 
Supported by National Key Research and Development Program of China (no. 2018YFC1314100) awarded to Yanbing Li; National Natural Science Foundation of China awarded to Yanbing Li (no. 81870557), Yan Guo (no. 82200876), and Hongyu Guan (no. 81572624); Key Field Research and Development Program of Guangdong Province, China (no. 2019B020230001) awarded to Yanbing Li; Guangdong Basic and Applied Basic Research Foundation (no. 2020A1515010049) awarded to Hai Li. 
Authorship: Yan Guo was responsible for the conceptualization, methodology, investigation, data curation, writing of the original draft preparation, and funding acquisition. Yanglei Cheng was responsible for the methodology, investigation, and resources. Hai Li was responsible for the data curation, funding acquisition, and resources. Hongyu Guan was responsible for the formal analysis and funding acquisition. Haipeng Xiao was responsible for the writing, reviewing, and editing, and resources. Yanbing Li was reponsbible for the conceptualization, writing, reviewing, and editing, and Supervision. 
Data Availability: The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request. 
Disclosure: Y. Gao, None; Y. Cheng, None; H. Li, None; H. Guan, None; H. Xiao, None; Y. Li, None 
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Figure 1.
 
Isolation and identification of OFs. (A) OFs were obtained from stromal vascular fraction (SVF) of orbital adipose tissues after collagenase digestion. (B) The surface marker expressions of TED-OFs (CD44, CD73, CD90, and CD105), hematopoietic cells (CD45), T lymphocytes (HLA-DR), B lymphocytes (CD19), and macrophages (CD11b) were analyzed by flow cytometry. (C) Relative microscopic images of TED-OFs of the indicated passages.
Figure 1.
 
Isolation and identification of OFs. (A) OFs were obtained from stromal vascular fraction (SVF) of orbital adipose tissues after collagenase digestion. (B) The surface marker expressions of TED-OFs (CD44, CD73, CD90, and CD105), hematopoietic cells (CD45), T lymphocytes (HLA-DR), B lymphocytes (CD19), and macrophages (CD11b) were analyzed by flow cytometry. (C) Relative microscopic images of TED-OFs of the indicated passages.
Figure 2.
 
Cellular cytotoxicity of ARS and the derivatives at different concentrations was tested by CCK-8 assays. (A) Chemical structures of three artemisinin-based drugs (ARSs). (B–D) CCK-8 assays were conducted to analyze cell cytotoxicity of TED-OFs treated with different concentration gradients of (B) ARS (0, 10, 25, 50, 100, 200, 500, and 1000 µM), (C) DHA (0, 0.5, 1, 5, 10, 20, 50, and 100 µM), and (D) ART (0, 0.5, 1, 5, 10, 20, 50, and 100 µM) for 24, 48, and 72 hours, respectively (n = 6 per group). (E) OFs from non-TED patients (non-TED-OFs) were treated with of ARS (200 µM), DHA (20 µM), and ART (10 µM) for 24, 48, and 72 hours, respectively (n = 6 per group). Relative CCK-8 intensity is presented as the percentage normalized to the average OD value of the DMSO-treated wells. The bar graph data are shown as the mean ± SEM from 6 independent TED-OF samples. *P < 0.05, **P < 0.01, ***P < 0.001, nsP ≥ 0.05 versus DMSO group.
Figure 2.
 
Cellular cytotoxicity of ARS and the derivatives at different concentrations was tested by CCK-8 assays. (A) Chemical structures of three artemisinin-based drugs (ARSs). (B–D) CCK-8 assays were conducted to analyze cell cytotoxicity of TED-OFs treated with different concentration gradients of (B) ARS (0, 10, 25, 50, 100, 200, 500, and 1000 µM), (C) DHA (0, 0.5, 1, 5, 10, 20, 50, and 100 µM), and (D) ART (0, 0.5, 1, 5, 10, 20, 50, and 100 µM) for 24, 48, and 72 hours, respectively (n = 6 per group). (E) OFs from non-TED patients (non-TED-OFs) were treated with of ARS (200 µM), DHA (20 µM), and ART (10 µM) for 24, 48, and 72 hours, respectively (n = 6 per group). Relative CCK-8 intensity is presented as the percentage normalized to the average OD value of the DMSO-treated wells. The bar graph data are shown as the mean ± SEM from 6 independent TED-OF samples. *P < 0.05, **P < 0.01, ***P < 0.001, nsP ≥ 0.05 versus DMSO group.
Figure 3.
 
Effects of ARS and the derivatives on adipogenesis of OFs in vitro. Forty-eight hours after growth arrest, the confluent TED-OFs were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with differentiation media (DM) containing ARSs at the indicated concentrations for 10 days. (A) Representative microscopic images of Oil Red O (ORO) staining in adipogenic OFs from patients with TED and non-TED patients. Scale bars = 400 µm. Similar images were obtained from six independent TED-OF samples. (B) Quantification of the ORO staining for TED-OFs in (A), as measured by the OD values (450 nm) after stained cells were solubilized (n = 6 per group). (C) Quantification of the ORO staining for non-TED-OFs in (A), as measured by the OD values (450 nm) after stained cells were solubilized (n = 6 per group). (D) Representative immunoblots with the adipogenesis markers in TED-OFs was assessed by Western blot analysis. GAPDH served as the control. Similar images were obtained from four independent TED-OF samples. (E–G) The protein levels were quantified, normalized to the level of GAPDH for each sample and analyzed (n = 4 per group). For (B), (C), and (E–G), the results are shown as the mean ± SEM. *P < 0.05, **P < 0.01. ***P < 0.001, nsP ≥ 0.05 versus the DMSO group.
Figure 3.
 
Effects of ARS and the derivatives on adipogenesis of OFs in vitro. Forty-eight hours after growth arrest, the confluent TED-OFs were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with differentiation media (DM) containing ARSs at the indicated concentrations for 10 days. (A) Representative microscopic images of Oil Red O (ORO) staining in adipogenic OFs from patients with TED and non-TED patients. Scale bars = 400 µm. Similar images were obtained from six independent TED-OF samples. (B) Quantification of the ORO staining for TED-OFs in (A), as measured by the OD values (450 nm) after stained cells were solubilized (n = 6 per group). (C) Quantification of the ORO staining for non-TED-OFs in (A), as measured by the OD values (450 nm) after stained cells were solubilized (n = 6 per group). (D) Representative immunoblots with the adipogenesis markers in TED-OFs was assessed by Western blot analysis. GAPDH served as the control. Similar images were obtained from four independent TED-OF samples. (E–G) The protein levels were quantified, normalized to the level of GAPDH for each sample and analyzed (n = 4 per group). For (B), (C), and (E–G), the results are shown as the mean ± SEM. *P < 0.05, **P < 0.01. ***P < 0.001, nsP ≥ 0.05 versus the DMSO group.
Figure 4.
 
Effects of ARS and the derivatives on the cellular proliferation of OFs in DM. Forty-eight hours after growth arrest, the confluent OFs (from patients with TED or non-TED patients) were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with DM for 20 hours. (A) Cartogram about cell cycle distribution of each TED-OFs group treated with or without ARS, DHA, and ART at the indicated concentrations in DM (n = 5 per group). (B) Representative experimental diagrams of the cell cycle distribution of each TED-OFs group treated with DMSO, ARS (200 µM), DHA (20 µM), and ART (10 µM) in DM, as determined by flow cytometry. (C) Representative images of the EdU incorporation assay results in each group of TED- and non-TED-OFs treated with DMSO, ARS (200 µM), DHA (20 µM), and ART (10 µM) in DM, scale bars = 100 µm. Red: EdU, Blue: DAPI. (D) Quantification of the EdU incorporation assays for (C), n = 5 per group. For (A) and (D), the data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, nsP ≥ 0.05 versus the DMSO group.
Figure 4.
 
Effects of ARS and the derivatives on the cellular proliferation of OFs in DM. Forty-eight hours after growth arrest, the confluent OFs (from patients with TED or non-TED patients) were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with DM for 20 hours. (A) Cartogram about cell cycle distribution of each TED-OFs group treated with or without ARS, DHA, and ART at the indicated concentrations in DM (n = 5 per group). (B) Representative experimental diagrams of the cell cycle distribution of each TED-OFs group treated with DMSO, ARS (200 µM), DHA (20 µM), and ART (10 µM) in DM, as determined by flow cytometry. (C) Representative images of the EdU incorporation assay results in each group of TED- and non-TED-OFs treated with DMSO, ARS (200 µM), DHA (20 µM), and ART (10 µM) in DM, scale bars = 100 µm. Red: EdU, Blue: DAPI. (D) Quantification of the EdU incorporation assays for (C), n = 5 per group. For (A) and (D), the data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, nsP ≥ 0.05 versus the DMSO group.
Figure 5.
 
Effects of ARS and the derivatives on the HA production of OFs in DM. Forty-eight hours after growth arrest, the confluent OFs (from patients with TED or non-TED patients) were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with DM for 48 hours, and then the supernatants were collected for ELISA analysis. (A) HA secretion in each TED (n = 5) and non-TED (n = 4) group were determined by ELISA. (B–F) The mRNA levels of HAS1 (B), HAS2 (C), HAS3 (D), HYAL1 (E), and HYAL2 (F) relative to those of GAPDH (normalized to the average values of the DMSO group), detected by qPCR in TED-OFs treated with or without ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 96 hours in DM. (B: n = 5; C–F: n = 3). The data are shown as the mean ± SEM, *P < 0.05, **P < 0.01, nsP ≥ 0.05 versus the DMSO group.
Figure 5.
 
Effects of ARS and the derivatives on the HA production of OFs in DM. Forty-eight hours after growth arrest, the confluent OFs (from patients with TED or non-TED patients) were pretreated by ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 24 hours, before being added with DM for 48 hours, and then the supernatants were collected for ELISA analysis. (A) HA secretion in each TED (n = 5) and non-TED (n = 4) group were determined by ELISA. (B–F) The mRNA levels of HAS1 (B), HAS2 (C), HAS3 (D), HYAL1 (E), and HYAL2 (F) relative to those of GAPDH (normalized to the average values of the DMSO group), detected by qPCR in TED-OFs treated with or without ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 96 hours in DM. (B: n = 5; C–F: n = 3). The data are shown as the mean ± SEM, *P < 0.05, **P < 0.01, nsP ≥ 0.05 versus the DMSO group.
Figure 6.
 
The mechanisms underlying in the effects of ARSs on the adipogenic TED-OFs. For (A–D) 48 hours after growth arrest, the confluent TED-OFs (n = 3) were pretreated by DMSO or ARS (200 µM) for 24 hours, before being added with DM for 96 hours. (A) Heat map of the DEGs in ARS versus DMSO. (B) KEGG pathway enrichment analysis for DEGs in ARS versus DMSO. (C) PANTHER pathway enrichment analysis for DEGs in ARS versus DMSO. (D) Venn diagram comparing the oppositely regulated DEGs in DM versus PM and ARS versus DMSO. (E) The mRNA levels of IGF1R (n = 3) and SCD (n = 3) relative to those of GAPDH (normalized to the average values of the DMSO group), detected by qPCR in TED-OFs treated with or without ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 96 hours in DM. Values are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the DMSO group. (F) Representative immunoblots with AKT and FOXO1 (phosphorylated and total) in TED- and non-TED-OFs was assessed by Western blot analysis. GAPDH served as the control. Similar images were obtained from three independent OF samples.
Figure 6.
 
The mechanisms underlying in the effects of ARSs on the adipogenic TED-OFs. For (A–D) 48 hours after growth arrest, the confluent TED-OFs (n = 3) were pretreated by DMSO or ARS (200 µM) for 24 hours, before being added with DM for 96 hours. (A) Heat map of the DEGs in ARS versus DMSO. (B) KEGG pathway enrichment analysis for DEGs in ARS versus DMSO. (C) PANTHER pathway enrichment analysis for DEGs in ARS versus DMSO. (D) Venn diagram comparing the oppositely regulated DEGs in DM versus PM and ARS versus DMSO. (E) The mRNA levels of IGF1R (n = 3) and SCD (n = 3) relative to those of GAPDH (normalized to the average values of the DMSO group), detected by qPCR in TED-OFs treated with or without ARS (0, 10, 50, and 200 µM), DHA (0, 1, 5, and 20 µM), or ART (0, 0.5, 2, and 10 µM) for 96 hours in DM. Values are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the DMSO group. (F) Representative immunoblots with AKT and FOXO1 (phosphorylated and total) in TED- and non-TED-OFs was assessed by Western blot analysis. GAPDH served as the control. Similar images were obtained from three independent OF samples.
Table 1.
 
Clinical Characteristics of Patients With TED in the Study
Table 1.
 
Clinical Characteristics of Patients With TED in the Study
Table 2.
 
Clinical Characteristics of Patients Without TED in the Study
Table 2.
 
Clinical Characteristics of Patients Without TED in the Study
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