Biochemistry and Molecular Biology Therapeutic Effect of Guggulsterone in Primary Cultured Orbital Fibroblasts Obtained From Patients with Graves’ Orbitopathy Bo Ram Kim,1 Jinjoo Kim,1 Jong Eun Lee,2 Eun Jig Lee,3 and Jin Sook Yoon1 1Department of Ophthalmology, Institute of Vision Research, Yonsei University of Medicine, Seoul, Korea 2Kyung Hee University College of Medicine, Seoul, Korea 3Department of Endocrinology, Severance Hospital, Yonsei University of Medicine, Seoul, Korea Correspondence: Jin Sook Yoon, PURPOSE. Inflammation, hyaluronan production, and adipogenesis are the main patho- Department of Ophthalmology, logical events leading to Graves’ orbitopathy (GO). Guggulsterone (GS), a phytosterol Severance Hospital, Institute of found in the resin of the guggul plant, is a well-known treatment for several inflamma- Vision Research, Yonsei University tory disorders, such as arthritis, obesity, and hyperlipidemia. Here we investigated the College of Medicine, 50-1 Yonsei-ro, effects of GS treatment on GO pathology. Seodaemun-gu, Seoul 03722, Korea; [email protected]. METHODS. Using primary cultures of orbital fibroblasts from GO patients and non-GO controls, we examined the effects of GS on hyaluronan production and the production Received: October 9, 2019 β Accepted: January 16, 2020 of proinflammatory cytokines induced by interleukin (IL)-1 , using real-time reverse Published: March 20, 2020 transcription-polymerase chain reaction analysis, western blots, and enzyme-linked immunosorbent assays. Further, adipogenic differentiation was evaluated by quantifi- Citation: Kim BR, Kim J, Lee JE, Lee cation of Oil Red O staining and assessment of protein levels of peroxisome proliferator EJ, Yoon JS. Therapeutic effect of γ α β guggulsterone in primary cultured activator gamma (PPAR ), CCAAT-enhancer-binding proteins (C/EBP) and ,andsterol orbital fibroblasts obtained from regulatory element-binding protein-1 (SREBP-1). patients with Graves’ orbitopathy. RESULTS. Treatment with noncytotoxic concentrations of GS resulted in the dose- Invest Ophthalmol Vis dependent inhibition of IL-1β-induced inflammatory cytokines, including IL-6, IL-8, MCP- Sci. 2020;61(3):39. https://doi.org/10.1167/iovs.61.3.39 1, and COX-2, at both mRNA and protein levels. The hyaluronan level was also signif- icantly suppressed by GS. Moreover, GS significantly decreased the formation of lipid droplets and expression of PPARγ ,C/EBPα/β, and SREBP-1 in a dose-dependent manner. GS pretreatment attenuated the phosphorylation of nuclear factor-kappa B induced by IL-1β. CONCLUSIONS. Our data show significant inhibitory effects of GS on inflammation, produc- tion of hyaluronan, and adipogenesis in orbital fibroblasts. To our knowledge, this is the first in vitro preclinical evidence of the therapeutic effect of GSinGO. Keywords: Graves’ orbitopathy, guggulsterone, inflammation, hyaluronan, adipogenesis raves’ orbitopathy (GO) is an autoimmune inflam- susceptibility to infection and adrenal insufficiency. More- G matory manifestation of Graves’ hyperthyroidism.1 over, they do not treat symptoms, such as proptosis, eyelid Its clinical manifestations are largely variable, including edema, and diplopia, in a large proportion of GO patients. severe pain, ocular discomfort, retraction of the eyelid, Despite the remarkable advances in drug development for ocular protrusion, and periorbital edema.2 Activation of the chronic autoimmune diseases, major drawbacks of the drugs thyroid-stimulating hormone receptor (TSHR) and insulin- for the treatment of GO are observed owing to an incomplete like growth factor 1 receptor, colocated in thyroid follicular understanding of its pathophysiology. cells and orbital fibroblasts, mediate the production of proin- Guggulsterone (GS) [4,17(20)-pregnadiene-3,16-dione], flammatory cytokines and chemokines. These cause massive a phytosterol present in the natural product gugulipid, infiltration of T and B cells, excessive hyaluronan accumu- which is derived from gum resin (Commiphora mukul), lation, and orbital adipogenesis, inducing globe proptosis.1 has been used as an effective herbal medicine to treat GO is difficult to manage owing to its diverse clini- atherosclerosis, rheumatism, hyperlipidemia, obesity, and cal progression and multiple causative factors. High-dose cancer.5–10 C. mukul has been shown to have a hypolipi- glucocorticoids have been the primary treatment option in demic effect through lowering total and LDL cholesterol active inflammatory states3; however, they are associated levels and triglycerides. It has also recently been used with several limitations, namely side effects, such as Cushing as a dietary supplement to lower cholesterol and treat syndrome, diabetes, hypertension, osteoporosis, and liver arthritis in the United States. GS was identified as the failure.4 Additionally, glucocorticoids might cause increased main bioactive constituent responsible for the therapeutic Copyright 2020 The Authors iovs.arvojournals.org | ISSN: 1552-5783 1 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 09/28/2021 Therapeutic Effect of Guggulsterone in GO IOVS | March 2020 | Vol. 61 | No. 3 | Article 39 | 2 TABLE. Clinical Information of Patient Samples Used for all In Vitro Studies Age (y) Sex CAS Smoker Duration of GO (y) Proptosis R/L (mm) Surgery Performed GO patients 44 F 0/7 N 11 21/21 Decompression 61 F 1/7 N 2 23/23 Decompression 45 F 0/7 N 4 26/26 Decompression 69 M 1/7 Y 1.8 19/19 Decompression 21 F 0/7 N 8 22/22 Decompression 29 M 0/7 Y 4 23.5/23.5 Decompression 54 M 4/7 Y 0.5 18/19 Decompression Non-GO control subjects 23 M N/A N N/A N/A Evisceration 75 M N/A N N/A N/A Evisceration 35 F N/A Y N/A N/A Orbital wall fracture 44 F N/A N N/A N/A Orbital wall fracture 47 F N/A N N/A N/A Orbital wall fracture CAS, clinical activity scores; Y, yes; N, No; N/A, not applicable; F, female; M, male; R, right eye; L, left eye. effects of C. mukul.8 Evidence has shown that the poten- between the third and fifth passages were used for experi- tial anti-inflammatory effects of GS are mediated by the ments. suppression of the nuclear factor-kappa B (NF-κB) signal- ing pathway and/or production of its dependent inflamma- tory cytokines. Additionally, several studies have shown that Reagents GS inhibits adipocyte differentiation, induces lipolysis in E-GS was obtained from Sigma–Aldrich, Inc. (St. Louis, 11 9 mature adipocytes, suppresses LDL oxidation, and acti- MO, USA). Oil Red O was purchased from Sigma–Aldrich, 12 vates adipocyte beiging. Here we aimed to explore the Inc. DMEM, penicillin, gentamicin, and FBS were acquired potential regulatory effect of GS with regard to the main from Hyclone Laboratories, Inc. (Logan, UT, USA). Recom- pathological events of GO in primary cultures of orbital binant human interleukin (IL)-1β was obtained from R&D fibroblasts from GO patients. Systems (Minneapolis, MN, USA). Anti-CCAAT-enhancer- binding protein (C/EBP) α and β, anti-sterol regula- tory element-binding protein-1 (SREBP-1), anti-peroxisome γ β MATERIALS AND METHODS proliferator activator gamma (PPAR ), and anti- -actin anti- bodies were purchased from Santa Cruz Biotechnology Cell Culture (Santa Cruz, CA, USA). Antibodies against extracellular signal-regulated kinase (ERK), phosphorylated ERK, Akt, Orbital tissue specimens were collected from the surgical phosphorylated Akt, c-Jun NH(2)-terminal kinase (JNK), waste of GO patients during orbital decompression surgery phosphorylated JNK, NF-κB, phosphorylated NF-κB, p38, for exophthalmos (n = 7; 3 men, 4 women; aged 21–69 and phosphorylated p38 were all purchased from Cell years). Healthy non-GO orbital tissue samples were collected Signaling Technology (Beverly, MA, USA). Phosphorylated during orbital wall fracture reduction or evisceration from levels of proteins were normalized to their respective total patients without a history or clinical evidence of any thyroid protein levels for each phosphorylation analysis. disease (n = 5; 2 men, 3 women; aged 23–75 years) (Table). All recruitment was done at the Department of Ophthalmol- ogy, Severance Hospital, Seoul, Korea. Healthy controls were Cell Viability also age- and sex-matched. The study protocol was approved by the institutional review board of Severance Hospital, Orbital fibroblast viability was examined by assessing 3-(4,5- Yonsei University College of Medicine, Seoul, Korea (IRB 4- dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide 2019-0397), and complied with the guidelines of the Decla- (MTT) reduction to formazan. Post-GS treatment at different ration of Helsinki. Written informed consent was obtained concentrations (5–50 μM) for 24 and 48 hours, both GO and from all participants. All GO patients were in a euthyroid non-GO cells were washed twice with PBS and incubated state for a minimum of 3 months presurgery with a ≤3 clin- for 3 hours with 5 mg/mL MTT solution at 37°C. After ical activity score at surgery.13 None of the GO patients had solubilization of the converted dye with dimethyl sulfoxide, received radiotherapy or high-dose steroid treatment for a absorbance was measured at 560 nm using a microplate minimum of 3 months prior to surgery. reader (EL 340 Bio Kinetics Reader; Bio-Tek Instruments, Primary cultures of orbital fibroblasts were established Winooski, VT, USA), with background subtraction at 630 nm. as previously described.14 Minced tissue explants were placed in plastic culture dishes, with penicillin (100 U/mL), Quantitative Real-Time PCR gentamycin (20 μg/mL), and Dulbecco’s modified Eagle’s medium (DMEM) containing 20% fetal bovine serum (FBS), Total RNA was extracted using TRIzol reagent (Invitrogen, to promote fibroblast proliferation. The cells were main- Carlsbad, CA, USA); 1 μg of RNA was used for first-strand tained in two 80-mm flasks containing DMEM, antibi- cDNA synthesis, according to the manufacturer’s protocol otics, and 10% FBS. Monolayers, treated with trypsin/EDTA, (#639543; Takara Bio, Inc., Shiga, Japan).