August 2016
Volume 57, Issue 10
Open Access
Biochemistry and Molecular Biology  |   August 2016
Therapeutic Effect of Protocatechuic Aldehyde in an In Vitro Model of Graves' Orbitopathy
Author Affiliations & Notes
  • Jung Woo Byun
    Department of Internal Medicine Institute of Endocrine Research, Yonsei University College of Medicine, Seoul, Republic of Korea
    Brain Korea 21 Plus Project for Medical Science, Yonsei University, Seoul, Korea
  • Sena Hwang
    Department of Internal Medicine Institute of Endocrine Research, Yonsei University College of Medicine, Seoul, Republic of Korea
  • Chan Woo Kang
    Brain Korea 21 Plus Project for Medical Science, Yonsei University, Seoul, Korea
  • Jin Hee Kim
    Department of Internal Medicine Institute of Endocrine Research, Yonsei University College of Medicine, Seoul, Republic of Korea
  • Min Kyung Chae
    Departments of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea
  • Jin Sook Yoon
    Departments of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea
  • Eun Jig Lee
    Department of Internal Medicine Institute of Endocrine Research, Yonsei University College of Medicine, Seoul, Republic of Korea
  • Correspondence: Eun Jig Lee, Department of Internal Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea; ejlee423@yuhs.ac
Investigative Ophthalmology & Visual Science August 2016, Vol.57, 4055-4062. doi:10.1167/iovs.15-19037
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      Jung Woo Byun, Sena Hwang, Chan Woo Kang, Jin Hee Kim, Min Kyung Chae, Jin Sook Yoon, Eun Jig Lee; Therapeutic Effect of Protocatechuic Aldehyde in an In Vitro Model of Graves' Orbitopathy. Invest. Ophthalmol. Vis. Sci. 2016;57(10):4055-4062. doi: 10.1167/iovs.15-19037.

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

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Abstract

Purpose: Protocatechuic aldehyde (3,4-dihydroxybenzaldehyde; PCA) is extracted from Salvia miltiorrhiza, and has been reported to possess antiproliferative, antioxidant, and antiadipogenesis properties in various in vivo and in vitro experiments. This study aimed to outline the antioxidant and suppressive effects of PCA on adipogenesis and hyaluronan production in orbital fibroblasts to help with designing therapeutic approaches for Graves' orbitopathy (GO).

Methods: We assessed the in vitro effects of PCA on orbital fibroblasts, which were cultured from orbital fat tissue obtained from patients undergoing orbital decompression for severe GO. Control tissue was obtained from patients undergoing orbital surgery with no history of GO or Graves' hyperthyroidism.

Results: The 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt assay results confirmed the free radical scavenging effect of PCA after treatment. Protocatechuic aldehyde exhibited a suppressive effect on intracellular reactive oxygen species generation and upregulated heme oxygenase-1 expression in Western blot analysis. Protocatechuic aldehyde attenuated TNF-α and IL-1β–induced hyaluronan production. Oil Red-O staining results revealed a decrease in lipid droplets and suppressed expression of the adipogenesis-related proteins peroxisome proliferator-activated receptor (PPAR)–γ, CCAAT/enhancer binding protein (c/EBP)-α, and c/EBP-β upon treatment with PCA during adipose differentiation.

Conclusions: In this study, PCA exerted significant antioxidant and antiadipogenic effects and inhibited the production of hyaluronan in GO orbital fibroblasts. Accordingly, PCA potentially could be used as a novel treatment option for GO.

Graves' disease (GD) is an autoimmune disorder that results from the excessive secretion of thyroid hormone stimulated by the binding of autoantibodies to thyroid hormone receptors expressed on thyroid follicular cells. In approximately 50% of GD patients, pathologic symptoms of the eyes, referred to as Graves' orbitopathy (GO), are observed.13 The general symptoms of GO include redness and retraction of the eyelids, along with edema of ocular tissue and exophthalmos. In most cases, GO development is characterized by acute inflammation that increases the volume of orbital connective/fatty tissue inside the orbital bone, leading to exophthalmos accompanied by severe pain.4,5 Increases in the volume of orbital tissue are characterized by the infiltration of immunocompetent cells, mainly T- and B-lymphocytes and mast cells,5,6 and stimulation of proinflammatory cytokine production in orbital fibroblasts leading to hyaluronan accumulation and adipogenesis. 
Although the specific mechanisms underlying the development of GO are unclear, oxygen free radicals are considered a possible factor.7 Upon increased optical oxidative stress, excessive adipogenesis and fibrosis can occur. Similarly, smokers, who possess higher levels of oxidative stress than nonsmokers, reportedly show more invasive GO.810 Recent studies also have found that not only do increases in oxidative stress induce adipogenesis, but they also increase the expressions of adipogenesis-related proteins.11 
Unfortunately, to date, there is no reliable or specific treatment for GO. Glucocorticoids, which comprise a class of steroid hormones secreted by the adrenal cortex, have been used as a first-line treatment for GO. However, glucocorticoid therapy can cause long-term side effects, such as osteoporosis, diabetes, depression, and Cushingoid features.12 Recently, attention has been focused on the antioxidant constituents in natural compounds, as they may be useful in the development of novel therapeutic medicines. Salvia miltiorrhiza, also known as red sage or danshen, is a perennial plant of the genus Salvia.13 The plant exhibits therapeutic effects on blood stasis, high blood pressure, and sclerogenic cardiomyopathy: in China, it has been used clinically to treat angina pectoris, hyperlipidemia, and acute ischemic stroke.1416 
Protocatechuic aldehyde (3,4-dihydroxybenzaldehyde; PCA) is the active compound extracted from Salvia miltiorrhiza, and has been reported to possess anti-inflammatory, antiproliferative, and antioxidant properties in various in vivo and in vitro experiments.1723 Recent studies have confirmed that PCA inhibits adipocyte differentiation and downregulates the expression of adipogenic transcription factors, such as peroxisome proliferator-activated–γ (PPAR-γ) receptor, CCAAT/enhancer binding protein–α (c/EBP-α), and the transcription factor sterol regulatory element-binding protein-1 (SREBP-1), in 3T3-L1 cells.24 Accordingly, this study was designed to determine the antioxidant and suppressive effects of PCA on adipogenesis and hyaluronan production in orbital fibroblasts and to determine the potential of using PCA for treating GO. 
Materials and Methods
Reagents and Chemicals
High glucose Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), gentamicin solution, and penicillin-streptomycin solution were purchased from GE Healthcare Life Sciences, Inc. (Logan, UT, USA). Protocatechuic aldehyde (3,4-dihydroxybenzaldehyde), Oil Red-O powder, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) powder and 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) powder were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay solution was purchased from Promega Corporation (Madison, WI, USA). The 5-(and-6)-carboxy-2′,7′-difluorodihydrofluorescein diacetate (Carboxy-H2DFFDA) was purchased from Invitrogen Molecular Probes, Inc. (Willow Creek Road, Eugene, OR, USA). The hyaluronan ELISA (HA ELISA) kit was purchased from Echelon Biosciences, Inc. (Salt Lake, UT, USA). Anti-heme oxygenase-1 (HO-1), anti-PPAR-γ, anti-c/EBP-α, anti-c/EBP-β, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). 
Orbital Fibroblasts Cell Culture
Primary orbital fibroblasts cultures were prepared as reported previously.4,11 Cell monolayers were serially passaged by gentle treatment with trypsin/EDTA and the third and fifth passages of cells were used for experiments. Orbital connective tissue was obtained from seven patients with GO during decompression surgery. All patients had euthyroid status and inactive GO with a clinical activity score <4 at the time of surgery and had not been treated with steroid or radiation for at least 3 months. Normal control tissues were obtained during upper lid blepharoplasties from four individuals with no history of GO or autoimmune thyroid disease. The study protocol was approved by the institutional review board of Severance Hospital, and all patients provided informed consent. This study followed the tenets of the Declaration of Helsinki. Before incubation with TNF-α or hydrogen peroxide (H2O2), cultured orbital fibroblasts were pretreated with PCA and DMEM medium supplemented with 1% FBS to study the suppressive effect of PCA on inflammation, oxidative stress, and hyaluronan production. 
DPPH Radical Scavenging Activity Assay
2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity was evaluated in a DPPH assay. A 0.1 mM solution of DPPH was prepared in ethanol. Increasing concentrations of PCA (0, 10, 50, 100 μM) were added to a 48-well culture plate. The DPPH solution then was added and allowed to react for 30 minutes at room temperature in the dark. Absorbance was measured at 517 nm using an ELISA plate reader. 
ABTS Radical Scavenging Activity Assay
We performed ABTS radical scavenging activity using an ABTS assay. A solution of ABTS was prepared by reacting 7.4 mM ABTS with 2.6 mM potassium persulfate for 24 hours in the dark at room temperature. Before beginning the assay, ABTS solution was diluted with PBS to an absorbance of 0.75 ± 0.05 at 734 nm. Protocatechuic aldehyde was added to a 48-well culture plate in increasing concentrations (0, 10, 50, 100 μM). The ABTS solution then was added and allowed to react for 15 minutes at room temperature in the dark. Absorbance was measured at 734 nm using an ELISA plate reader. 
Cell Viability (MTS) Assay
Cell viability was assessed using the MTS assay according to the manufacturer's specifications. Orbital fibroblasts cells were seeded into 96-well culture plates (1 × 104 cells/well). Serial concentrations of PCA (0, 10, 30, 50, 100, 200 μM) were added to the wells, and the plate was incubated at 37°C and 5% CO2 for 24, 48, or 72 hours. Thereafter, MTS solution was added and the plate was incubated again for 4 hours under the same conditions. Absorbance of the dye was measured at 490 nm using an ELISA plate reader. 
Measurement of Intracellular Reactive Oxygen Species (ROS) Generation
Reactive oxygen species generation was detected with Carboxy-H2DFFDA. Cells were treated with serial concentrations of PCA (0, 10, 50, 100 μM), and incubated at 37°C and 5% CO2 for 24 hours. After 24 hours, the cells were washed with Dulbecco's phosphate buffered saline (DPBS), incubated with 10 μM Carboxy-H2DFFDA for 30 minutes, and stimulated with H2O2 (100 μM) for 30 minutes. Subsequent to H2O2 stimulation, the cells were trypsinized and washed in cold DPBS. Fluorescence intensity was measured with flow cytometry (FACSverse; BD Biosciences, Franklin Lakes, NJ, USA), and a flow cytometric analysis was performed using the Flow JO software (TreeStar, Inc., Ashland, OR, USA). 
Western Blot Assay
First, cells were washed with cold DPBS and incubated in lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 mM NaF, 10% glycerol, 20 mM HEPES (pH 7.2), 0.1 mM dithiothreitol, 1 mg/mL pepstatin, 1 mg/mL leupeptin, 10 mM Na3VO4, and 1% Triton X-100 for 30 minutes on ice. Lysates then were centrifuged for 20 minutes at 13,000g. Protein concentrations were determined by the Quick Start Bradford Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The same quantities of protein were boiled at 95°C for 5 minutes with the sample buffer, and resolved on 8% or 10% (wt/vol) SDS-PAGE gels. Protein bands were transferred onto polyvinylidene fluoride membranes (Immobilon; Millipore, Billerica, MA, USA) using the wet transfer method. Membranes were blocked with 5% skim milk in Tris-Buffered Saline Tween-20 (TBST) for 2 hours. After blocking, the blots were probed overnight with primary antibodies (HO-1, PPAR-γ, c/EBP-α, and β) in TBST containing 5% BSA at 4°C. After treatment with the secondary antibody, immunoreactive bands were detected with horseradish peroxidase conjugated and developed with an enhanced chemiluminescent Western blotting detection kit (GE Healthcare Life Sciences, Inc.). 
Hyaluronan ELISA
Orbital fibroblasts were seeded in a 96-well culture plate (1 × 104 cells/well) and incubated at 37°C in 5% CO2. After the cells reached confluence, the PCA-treated samples (0, 50, 100 μM) then were stimulated with 10 ng/mL TNF-α or 1 ng/mL IL-1β. Supernatant from the cell culture medium was collected. The hyaluronan concentration was determined using an HA ELISA kit. 
Adipocyte Differentiation
Adipocyte differentiation was induced as described previously.4,11 Cells were seeded in either a 6-cm or 6-well plate and reached a desired confluence. To induce adipocyte differentiation, the culture medium was replaced with DMEM supplemented with 10% FBS, 33 μM biotin, 17 μM pantothenic acid, 0.2 nM T3, 10 μg/mL transferrin, 0.2 μM prostaglandin I2, 0.1 mM isobutylmethylxanthine (IBMX), 1 μM dexamethasone (Sigma-Aldrich Corp.), 1 μM insulin (Roche Diagnostics Corporation, Indianapolis, IN, USA), and 10 μM of the PPAR agonist rosiglitazone (Cayman Chemical Company, Ann Arbor, MI, USA). The differentiation medium was replaced every 2 to 3 days for 10 days. For 4 days thereafter, differentiation medium lacking IBMX, dexamethasone, and insulin was used. 
Oil Red-O Staining
After 10 days of adipocyte differentiation, cells were stained with Oil Red O. A stock solution of Oil Red O (0.5% Oil Red O in isopropanol) was prepared. Working solution was prepared by diluting 6 mL of the stock solution in 4 mL distilled water. The resulting solution was allowed to mix for 30 minutes at 70°C and then was filtered twice. Cells were washed with DPBS and fixed with 10% formalin overnight at 4°C. After fixation, cells were washed with distilled water and stained with Oil Red O working solution for 15 to 20 minutes at room temperature. After staining, cells were washed with distilled water and kept at 4°C 
Statistical Analysis
All experiments were performed at least in triplicate. Data were analyzed by the Mann-Whitney U test or Student's t-test (SPSS for Windows version 20.0.0.2; SPSS, Inc., Chicago, IL, USA). All P values <0.05 were considered to be significant. 
Results
Effect of PCA on ROS Generation in GO Orbital Fibroblasts
To assess the free radical scavenging effects of PCA, DPPH (Fig. 1A) and ABTS assays (Fig. 1B) were performed with increasing concentrations of PCA (0, 10, 50, and 100 μM). As shown in Figures 1A and 1B, nearly all free radicals were scavenged at 50 μM in the DPPH and ABTS assays (94.3% ± 0.18%; P < 0.001; DPPH assay), (95.6% ± 0.5%; P < 0.001; ABTS assay). To assess the effect of PCA on ROS generation in GO orbital fibroblasts, FACS analysis was performed. Since PCA treatment (0–200 μM) did not affect the viability of GO orbital fibroblasts (Supplementary Fig. S1A), PCA concentrations of 0, 50, and 100 μM were used to determine its effects on ROS generation. First, however, GO orbital fibroblasts were stimulated with H2O2 (10, 50, and 100 μM) for 30 minutes to confirm ROS generation in the orbital fibroblasts, in which significant dose-dependent increases were observed (Supplementary Fig. S1B). As shown in Figure 1C, treatment with 50 and 100 μM of PCA for 24 hours significantly inhibited ROS generation in GO orbital fibroblasts (50 μM PCA, 89.8% ± 9.2%, P < 0.05; 100 μM PCA, 88.2% ± 13.9%, P < 0.05). Additionally, following pretreatment with PCA for 24 hours and stimulation with 100 μM H2O2 for 30 minutes, ROS generation decreased significantly (100 μM H2O2, 280.6% ± 45.7%, P < 0.001; 50 μM PCA, 215.4% ± 39.3%, P < 0.005; 100 μM PCA, 235.7% ± 48.0%, P < 0.05; Fig. 1D). 
Figure 1
 
Effect of PCA on ROS generation in GO orbital fibroblasts. Increasing concentrations of PCA (0, 10, 50, and 100 μM) were reacted with DPPH (A) and ABTS solution (B). To determine the effect of PCA on ROS generation, cells were pretreated with PCA (0, 50, and 100 μM) for 24 hours (C) and stimulated with 100 μM H2O2 for 30 minutes (D). Data represent the mean ± SD of three experiments. The results are expressed as a percentage of control values (*P < 0.05, ***P < 0.001 versus untreated control; †P < 0.05, ††P < 0.005 versus treated with 100 μM H2O2 alone).
Figure 1
 
Effect of PCA on ROS generation in GO orbital fibroblasts. Increasing concentrations of PCA (0, 10, 50, and 100 μM) were reacted with DPPH (A) and ABTS solution (B). To determine the effect of PCA on ROS generation, cells were pretreated with PCA (0, 50, and 100 μM) for 24 hours (C) and stimulated with 100 μM H2O2 for 30 minutes (D). Data represent the mean ± SD of three experiments. The results are expressed as a percentage of control values (*P < 0.05, ***P < 0.001 versus untreated control; †P < 0.05, ††P < 0.005 versus treated with 100 μM H2O2 alone).
Effect of PCA on HO-1 Expression in GO Orbital Fibroblasts
Treatment with 10, 50, and 100 μM of PCA for 24 hours significantly upregulated HO-1 protein expression (PCA 100 μM, 329.1% ± 137.4%, P < 0.05; Fig. 2A). Cells pretreated with increasing concentrations of PCA for 24 hours followed by stimulation with 100 μM H2O2 for 16 hours showed a dose-dependent increase in HO-1 protein expression (PCA 100 μM, 479.3% ± 57.7%, P < 0.005; Fig. 2B). When GO cells were preincubated for 1 hour with zinc protoporphyrin (ZnPP), an HO-1 inhibitor, PCA failed to prevent ROS generation in GO cells exposed to H2O2, indicating that the antioxidant effects of PCA likely rely on HO-1. Interestingly, when HO-1 was inhibited, H2O2 treatment further potentiated the increases in ROS generation (Fig. 2C). 
Figure 2
 
Western blot analysis of PCA effects on HO-1 expression in GO orbital fibroblasts. The cells were pretreated with increasing concentrations of PCA (0, 10, 50, and 100 μM) for 24 hours (A) and then stimulated with H2O2 100 μM for 16 hours (B). Anti-heme oxygenase-1 was quantified by densitometry and normalized to the level of β-actin in the same sample. To confirm the protective role of HO-1 against ROS generation in GO, the cells were pretreated with 5 μM ZnPP IX (an HO-1 inhibitor) for 1 hour, followed by 100 μM PCA treatment for 24 hours, and then stimulated with 100 μM H2O2 for 30 minutes. Data represent means ± SD of three experiments. The results are expressed as percentages of control values (*P < 0.05, ***P < 0.001 versus untreated control; ††P < 0.005, †††P < 0.001 versus treated with 100 μM H2O2 alone).
Figure 2
 
Western blot analysis of PCA effects on HO-1 expression in GO orbital fibroblasts. The cells were pretreated with increasing concentrations of PCA (0, 10, 50, and 100 μM) for 24 hours (A) and then stimulated with H2O2 100 μM for 16 hours (B). Anti-heme oxygenase-1 was quantified by densitometry and normalized to the level of β-actin in the same sample. To confirm the protective role of HO-1 against ROS generation in GO, the cells were pretreated with 5 μM ZnPP IX (an HO-1 inhibitor) for 1 hour, followed by 100 μM PCA treatment for 24 hours, and then stimulated with 100 μM H2O2 for 30 minutes. Data represent means ± SD of three experiments. The results are expressed as percentages of control values (*P < 0.05, ***P < 0.001 versus untreated control; ††P < 0.005, †††P < 0.001 versus treated with 100 μM H2O2 alone).
Effect of PCA on TNF-α– or IL-1β–Induced Hyaluronan Production in GO Orbital Fibroblasts
To assess the effects of PCA on TNF-α– or IL-1β–induced hyaluronan production, hyaluronan ELISA was performed. For hyaluronan ELISA analysis, cells were treated with increasing concentrations of PCA (0, 50, and 100 μM) for 24 hours following TNF-α or IL-1β stimulation. As shown in Figure 3A, hyaluronan production significantly increased following stimulation with 10 ng/mL TNF-α (119.5% ± 15.3%, P < 0.05) compared to untreated control cells; PCA decreased the hyaluronan production induced by TNF-α (50 μM PCA, 95.0% ± 15.5%, P < 0.05). As shown in Figure 3B, hyaluronan production significantly increased following stimulation with 1 ng/mL IL-1β (226.0% ± 34.4%, P < 0.001) compared to untreated control cells; PCA decreased the hyaluronan production induced by IL-1β in a dose-dependent manner (50 μM PCA, 179.9% ± 27.3%, P < 0.05; 100 μM PCA, 172.0% ± 27.1%, P < 0.005). 
Figure 3
 
Effect of PCA on hyaluronan production in GO orbital fibroblasts. Cells were pretreated with increasing concentrations of PCA (0, 50, and 100 μM) for 24 hours and stimulated with 10 ng/mL TNF-α (A) or 1 ng/mL IL-1β (B). Data represent the mean ± SD of three experiments (*P < 0.05, ***P < 0.001 versus untreated control; †P < 0.05, ††P < 0.005 versus treated with TNF-α or IL-1β alone).
Figure 3
 
Effect of PCA on hyaluronan production in GO orbital fibroblasts. Cells were pretreated with increasing concentrations of PCA (0, 50, and 100 μM) for 24 hours and stimulated with 10 ng/mL TNF-α (A) or 1 ng/mL IL-1β (B). Data represent the mean ± SD of three experiments (*P < 0.05, ***P < 0.001 versus untreated control; †P < 0.05, ††P < 0.005 versus treated with TNF-α or IL-1β alone).
Effects of PCA on Adipogenesis in GO Orbital Fibroblasts
To determine the effect of PCA on adipogenesis, increasing concentrations of PCA (0, 10, 50, and 100 μM) were added to an adipogenic medium containing 10 μM rosiglitazone. Oil Red-O staining and examination under a high-powered (×400) microscope revealed a significant reduction in the size and number of lipid droplets upon PCA treatment (Fig. 4A). Absorbance at 490 nm for PCA-treated cells was significantly reduced (Fig. 4B). Additionally, Western blot analysis results revealed that PCA attenuated the expressions of the adipogenic transcription factors PPAR-γ, c/EBP-α, and c/EBP-β during adipogenesis (Fig. 4C). 
Figure 4
 
Effect of PCA on adipogenesis in GO orbital fibroblasts. Cells were treated with PCA (0, 10, 50, and 100 μM) during the first 4 days of adipocyte differentiation. After differentiation, cells were stained with Oil Red-O to visualize lipid accumulation and examined microscopically (×400; A). Cell-bound Oil Red-O stain was solubilized and the optical density (OD) thereof was read at 490 nm to quantitatively assess adipogenesis (B). Cell lysates were subjected to Western blot analysis to assess PPAR-γ, c/EBP-α, and c/EBP-β protein expression; PPAR-γ, c/EBP-α, and c/EBP-β were quantified by densitometry and normalized to the level of β-actin in the same sample. Data are represented as mean relative density ratios ± SD of three experiments (*P < 0.05, **P < 0.005, ***P < 0.001 versus untreated control; C).
Figure 4
 
Effect of PCA on adipogenesis in GO orbital fibroblasts. Cells were treated with PCA (0, 10, 50, and 100 μM) during the first 4 days of adipocyte differentiation. After differentiation, cells were stained with Oil Red-O to visualize lipid accumulation and examined microscopically (×400; A). Cell-bound Oil Red-O stain was solubilized and the optical density (OD) thereof was read at 490 nm to quantitatively assess adipogenesis (B). Cell lysates were subjected to Western blot analysis to assess PPAR-γ, c/EBP-α, and c/EBP-β protein expression; PPAR-γ, c/EBP-α, and c/EBP-β were quantified by densitometry and normalized to the level of β-actin in the same sample. Data are represented as mean relative density ratios ± SD of three experiments (*P < 0.05, **P < 0.005, ***P < 0.001 versus untreated control; C).
Discussion
Graves' orbitopathy is characterized by autoimmune inflammation, which leads to thickening of the connective and adipose tissue within the bony orbit of the eye. This increase in tissue volume leads to the development of exophthalmos accompanied by severe pain. Currently, there is no reliable or specific treatment for GO, and researchers have turned to natural compounds in an effort to develop new treatments. The biologically active component PCA, a single elemental substance extracted from S. miltiorrhiza, has been reported to possess anti-inflammatory, antioxidant, and antiadipogenic properties in multiple in vivo and in vitro studies.1723 However, PCA has not been evaluated for its specific effects against GO in an in vitro model. In this study, we found that PCA significantly inhibited oxidative stress, production of hyaluronan, and adipogenesis in GO orbital fibroblasts. The inhibitory effect of PCA was attributable to drug cytotoxicity, as confirmed in the MTS assay. 
Oxidative stress is related to the development of GO. Recent studies have found that an increased oxidative stress induces greater levels of adipogenesis.11,2528 We confirmed in the DPPH and ABTS assays that PCA exhibits free radical scavenging activity. Therefore, we believe that the antioxidant effect of PCA probably is associated with its antiadipogenic effect. 
In this investigation, we demonstrated that PCA inhibits intracellular ROS generation. To determine the mechanism by which PCA functions to suppress ROS generation, we examined the effect of PCA on HO-1 expression by Western blot analysis. The antioxidant enzyme HO catalyzes the oxidative degradation of heme into biliverdin, carbon monoxide, and iron, and HO-1 is one of the three isoforms of HO activated in response to stress induced by hypoxia, heavy metals, and cytokines.29,30 Therefore, upregulation of HO-1 is considered to offer generalized cellular protection against oxidative stress, thereby acting as an active defense mechanism.31 Reportedly, oxidative stress stimulated ROS production dose dependently in GO-related orbital fibroblasts, and HO-1 expression was increased in the protective response to oxidative stress.32 We confirmed that the expression of HO-1 is upregulated in a dose-dependent manner in GO orbital fibroblasts treated with PCA. To identify the relative protective effect of HO-1 against ROS generation, the cells were additionally treated with ZnPP IX, an HO-1 inhibitor found in red blood cells when heme production is inhibited by a lead or by lack of iron.33 In the presence of ZnPP IX, the generation of intracellular ROS increased; this increase was not affected by subsequent treatment with PCA. Based on these results, the antioxidant effects of PCA are exerted through the upregulation of HO-1 protein expression, ultimately leading to ROS reduction in GO orbital fibroblasts. 
In the early phase of GO, T cells infiltrate and interact with orbital fibroblasts to promote cytokine production and the secretion of T-cell activating factors.6 Stimulation of these fibroblasts induces the release of multiple cytokines that stimulate B-cell differentiation and GD IgG.34 Reportedly, treatment with TNF-α and IL-1β increases in hyaluronan production.4,35 We confirmed that TNF-α or IL-1β increases hyaluronan production in GO orbital fibroblasts in this study, and this action was inhibited by treatment with PCA. 
As previously reported, PCA inhibits adipocyte differentiation and downregulates the expression of adipogenic transcription factors in 3T3-L1 cells.24 Our data also showed that PCA treatment suppresses adipogenesis in GO orbital fibroblasts in a dose-dependent manner. Microscopic examination following Oil Red-O staining showed that PCA inhibits the accumulation of lipid droplets. The expression of PPAR-γ, c/EBP-α, and c/EBP-β is related to adipogenesis differentiation, and PPAR-γ and c/EBP-α are expressed during phases of adipogenesis.36 In our study, PCA treatment resulted in reduced protein expression of PPAR-γ, c/EBP-α, and their upstream regulator, c/EBP-β. Therefore, we postulated that PCA exerts its antiadipogenic effects by suppressing these adipogenic transcription factors. 
In conclusion, we confirmed that PCA exhibits antioxidant and antiadipogenic properties. Our results suggested that underlying the antiadipogenic effect of PCA is associated with its antioxidant effect, which is related upregulation of HO-1 protein expression. Additionally, PCA showed an inhibitory effect on hyaluronan production. Taken together, we identified that PCA significantly inhibited all pathologic mechanisms associated with the development of GO. Therefore, PCA could potentially be used as a novel agent for the prevention and treatment of GO. Further research and clinical studies are required to confirm the safety of PCA. 
Acknowledgments
Supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (Grant 2014M3A9B6069341). 
Disclosure: J.W. Byun, None; S. Hwang, None; C.W. Kang, None; J.H. Kim, None; M.K. Chae, None; J.S. Yoon, None; E.J. Lee, None 
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Figure 1
 
Effect of PCA on ROS generation in GO orbital fibroblasts. Increasing concentrations of PCA (0, 10, 50, and 100 μM) were reacted with DPPH (A) and ABTS solution (B). To determine the effect of PCA on ROS generation, cells were pretreated with PCA (0, 50, and 100 μM) for 24 hours (C) and stimulated with 100 μM H2O2 for 30 minutes (D). Data represent the mean ± SD of three experiments. The results are expressed as a percentage of control values (*P < 0.05, ***P < 0.001 versus untreated control; †P < 0.05, ††P < 0.005 versus treated with 100 μM H2O2 alone).
Figure 1
 
Effect of PCA on ROS generation in GO orbital fibroblasts. Increasing concentrations of PCA (0, 10, 50, and 100 μM) were reacted with DPPH (A) and ABTS solution (B). To determine the effect of PCA on ROS generation, cells were pretreated with PCA (0, 50, and 100 μM) for 24 hours (C) and stimulated with 100 μM H2O2 for 30 minutes (D). Data represent the mean ± SD of three experiments. The results are expressed as a percentage of control values (*P < 0.05, ***P < 0.001 versus untreated control; †P < 0.05, ††P < 0.005 versus treated with 100 μM H2O2 alone).
Figure 2
 
Western blot analysis of PCA effects on HO-1 expression in GO orbital fibroblasts. The cells were pretreated with increasing concentrations of PCA (0, 10, 50, and 100 μM) for 24 hours (A) and then stimulated with H2O2 100 μM for 16 hours (B). Anti-heme oxygenase-1 was quantified by densitometry and normalized to the level of β-actin in the same sample. To confirm the protective role of HO-1 against ROS generation in GO, the cells were pretreated with 5 μM ZnPP IX (an HO-1 inhibitor) for 1 hour, followed by 100 μM PCA treatment for 24 hours, and then stimulated with 100 μM H2O2 for 30 minutes. Data represent means ± SD of three experiments. The results are expressed as percentages of control values (*P < 0.05, ***P < 0.001 versus untreated control; ††P < 0.005, †††P < 0.001 versus treated with 100 μM H2O2 alone).
Figure 2
 
Western blot analysis of PCA effects on HO-1 expression in GO orbital fibroblasts. The cells were pretreated with increasing concentrations of PCA (0, 10, 50, and 100 μM) for 24 hours (A) and then stimulated with H2O2 100 μM for 16 hours (B). Anti-heme oxygenase-1 was quantified by densitometry and normalized to the level of β-actin in the same sample. To confirm the protective role of HO-1 against ROS generation in GO, the cells were pretreated with 5 μM ZnPP IX (an HO-1 inhibitor) for 1 hour, followed by 100 μM PCA treatment for 24 hours, and then stimulated with 100 μM H2O2 for 30 minutes. Data represent means ± SD of three experiments. The results are expressed as percentages of control values (*P < 0.05, ***P < 0.001 versus untreated control; ††P < 0.005, †††P < 0.001 versus treated with 100 μM H2O2 alone).
Figure 3
 
Effect of PCA on hyaluronan production in GO orbital fibroblasts. Cells were pretreated with increasing concentrations of PCA (0, 50, and 100 μM) for 24 hours and stimulated with 10 ng/mL TNF-α (A) or 1 ng/mL IL-1β (B). Data represent the mean ± SD of three experiments (*P < 0.05, ***P < 0.001 versus untreated control; †P < 0.05, ††P < 0.005 versus treated with TNF-α or IL-1β alone).
Figure 3
 
Effect of PCA on hyaluronan production in GO orbital fibroblasts. Cells were pretreated with increasing concentrations of PCA (0, 50, and 100 μM) for 24 hours and stimulated with 10 ng/mL TNF-α (A) or 1 ng/mL IL-1β (B). Data represent the mean ± SD of three experiments (*P < 0.05, ***P < 0.001 versus untreated control; †P < 0.05, ††P < 0.005 versus treated with TNF-α or IL-1β alone).
Figure 4
 
Effect of PCA on adipogenesis in GO orbital fibroblasts. Cells were treated with PCA (0, 10, 50, and 100 μM) during the first 4 days of adipocyte differentiation. After differentiation, cells were stained with Oil Red-O to visualize lipid accumulation and examined microscopically (×400; A). Cell-bound Oil Red-O stain was solubilized and the optical density (OD) thereof was read at 490 nm to quantitatively assess adipogenesis (B). Cell lysates were subjected to Western blot analysis to assess PPAR-γ, c/EBP-α, and c/EBP-β protein expression; PPAR-γ, c/EBP-α, and c/EBP-β were quantified by densitometry and normalized to the level of β-actin in the same sample. Data are represented as mean relative density ratios ± SD of three experiments (*P < 0.05, **P < 0.005, ***P < 0.001 versus untreated control; C).
Figure 4
 
Effect of PCA on adipogenesis in GO orbital fibroblasts. Cells were treated with PCA (0, 10, 50, and 100 μM) during the first 4 days of adipocyte differentiation. After differentiation, cells were stained with Oil Red-O to visualize lipid accumulation and examined microscopically (×400; A). Cell-bound Oil Red-O stain was solubilized and the optical density (OD) thereof was read at 490 nm to quantitatively assess adipogenesis (B). Cell lysates were subjected to Western blot analysis to assess PPAR-γ, c/EBP-α, and c/EBP-β protein expression; PPAR-γ, c/EBP-α, and c/EBP-β were quantified by densitometry and normalized to the level of β-actin in the same sample. Data are represented as mean relative density ratios ± SD of three experiments (*P < 0.05, **P < 0.005, ***P < 0.001 versus untreated control; C).
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