Therapeutic Effect of Resveratrol on Oxidative Stress in Graves' Orbitopathy Orbital Fibroblasts

Kim, Chang Yeom; Lee, Hyun Jung; Chae, Min Kyung; Byun, Jung Woo; Lee, Mi Kyung; Yoon, Jin Sook

doi:10.1167/iovs.15-16870

Cited by 26 publications

(20 citation statements)

References 46 publications

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“…Previously, we have found that quercetin inhibits cigarette smoke extract-induced adipogenesis in GO fibroblasts by reducing ROS (48). Additionally, we have reported several molecules with anti-oxidative properties such as resveratrol, caffeine, and curcumin suppress adipogenesis in GO fibroblasts (49)(50)(51). Given that oxidative stress contributes to the proliferation of orbital fibroblasts (52), the impeded proliferation of GO fibroblasts by PCSK9 inhibition demonstrated in this study may be attributed to the anti-oxidative property of the PCSK9 inhibitor.…”

Section: Discussionmentioning

confidence: 97%

Role of Proprotein Convertase Subtilisin/Kexin Type 9 in the Pathogenesis of Graves’ Orbitopathy in Orbital Fibroblasts

et al. 2021

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BackgroundThe proprotein convertase subtilisin/kexin type 9 (PCSK9) has been implicated in the pathogenesis of inflammatory diseases. We sought to investigate the role of PCSK9 in the pathogenesis of Graves’ orbitopathy (GO) and whether it may be a legitimate target for treatment.MethodsThe PCSK9 was compared between GO (n=11) and normal subjects (n=7) in orbital tissue explants using quantitative real-time PCR, and in cultured interleukin-1β (IL-1β)-treated fibroblasts using western blot. Western blot was used to identify the effects of PCSK9 inhibition on IL-1β-induced pro-inflammatory cytokines production and signaling molecules expression as well as levels of adipogenic markers and oxidative stress-related proteins. Adipogenic differentiation was identified using Oil Red O staining. The plasma PCSK9 concentrations were compared between patients with GO (n=44) and healthy subjects (n=26) by ELISA.ResultsThe PCSK9 transcript level was higher in GO tissues. The depletion of PCSK9 blunted IL-1β-induced expression of intercellular adhesion molecule 1 (ICAM-1), IL-6, IL-8, and cyclooxygenase-2 (COX-2) in GO and non-GO fibroblasts. The levels of activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and phosphorylated forms of Akt and p38 were diminished when PCSK9 was suppressed in GO fibroblasts. Decreases in lipid droplets and attenuated levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein β (C/EBPβ), and leptin as well as hypoxia-inducible factor 1α (HIF-1α), manganese superoxide dismutase (MnSOD), thioredoxin (Trx), and heme oxygenase-1 (HO-1) were noted when PCSK9 was suppressed during adipocyte differentiation. The plasma PCSK9 level was significantly higher in GO patients and correlated with level of thyrotropin binding inhibitory immunoglobulin (TBII) and the clinical activity score (CAS).ConclusionsPCSK9 plays a significant role in GO. The PCSK9 inhibition attenuated the pro-inflammatory cytokines production, oxidative stress, and fibroblast differentiation into adipocytes. PCSK9 may serve as a therapeutic target and biomarker for GO.

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Section: Discussionmentioning

confidence: 97%

Role of Proprotein Convertase Subtilisin/Kexin Type 9 in the Pathogenesis of Graves’ Orbitopathy in Orbital Fibroblasts

et al. 2021

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“…Based on clinical reports, cigarette smoking-likely the most important environmental factor associated with GO occurrence and progressionmay act, among other mechanisms, by stimulating the generation of ROS and reducing antioxidant production [3,20,21]. In this regard, several in vitro studies demonstrate the therapeutic effectiveness of drugs with antioxidant potential in primary cultured orbital fibroblasts from GO patients [12,[15][16][17]. Clinical studies on the use of antioxidants for the management of GO have also shown them to be clinically effective in patients experiencing mild severity [22][23][24].…”

Section: Discussionmentioning

confidence: 99%

“…ROS release was determined with 5-(and 6)carboxy-20,70-dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen/Thermo Fisher Scientific, Carlsbad, CA, USA), an oxidant-sensitive fluorescent probe, as we described previously [12]. Briefly, primary cultured orbital fibroblasts were pretreated with various concentrations of caffeine for 24 h. The culture medium was then removed, and the cells were washed with PBS, incubated with 10 mM H2DCFDA at 37°C for 30 min, then stimulated with CSE (2%) or H 2 O 2 (10 μM) for 30 min [12,15,16]. The fluorescently stained cells were examined microscopically at 40× and were quantified via flow-cytometric analysis.…”

Section: Intracellular Ros Measurementmentioning

confidence: 99%

Anti-oxidative and anti-adipogenic effects of caffeine in an <i>in vitro</i> model of Graves’ orbitopathy

Kim

et al. 2020

Endocr J

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Oxidative stress and adipogenesis play key roles in the pathogenesis of Graves' orbitopathy (GO). In this study, the therapeutic effects of caffeine on the reduction of oxidative stress and adipogenesis were evaluated in primary cultured GO orbital fibroblasts in vitro. Orbital fibroblasts were cultured from orbital connective tissues obtained from individuals with GO. Intracellular reactive oxygen species (ROS) levels induced by hydrogen peroxide or cigarette smoke extract and the expression of anti-oxidative enzymes were measured after caffeine treatment. After adipogenic differentiation and caffeine treatment, cells were stained with Oil Red O and the levels of peroxisome proliferator activator γ (PPARγ), C/EBPα, and C/ EBPβ were determined by western blot analysis. Hydrogen peroxide and cigarette smoke extract increased the levels of intracellular ROS and anti-oxidative enzymes, which decreased in a dose-dependent manner upon pretreatment with caffeine in GO orbital fibroblasts. Oil Red-O staining results revealed a decrease in lipid droplets; furthermore, PPARγ, C/EBPα, and C/EBPβ protein expression levels were inhibited upon treatment with caffeine during adipocyte differentiation. In conclusion, caffeine decreased oxidative stress and adipogenesis in GO orbital fibroblasts in vitro. These findings may contribute to the development of new types of caffeine-containing pharmacological agents for use in the management of GO.

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“…Orbital fibroblasts subjected to different study conditions were washed with PBS and then lysed with cell lysis buffer on ice. 12 The lysates were centrifuged, and the supernatants were resolved in 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto polyvinylidene fluoride membranes (Immobilon; Millipore Corp., Billerica, MA, USA). The membranes were then probed with primary antibodies in tris-buffered saline and Tween 20 along with 1% BSA and 0.01% sodium azide.…”

Section: Methodsmentioning

confidence: 99%

Glycogen Synthase Kinase-3β Mediates Proinflammatory Cytokine Secretion and Adipogenesis in Orbital Fibroblasts from Patients with Graves’ Orbitopathy

Lee

Chae

Kikkawa

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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Purpose We sought to determine the role of glycogen synthase kinase-3β (GSK-3β) in the pathogenesis of Graves’ orbitopathy(GO). Methods Expression of the GSK-3β gene in whole orbital tissue explants was compared between GO and non-GO donors using quantitative real-time PCR (RT-PCR). The expression of proinflammatory molecules in the presence of the GSK-3β inhibitor CHIR 99021 was analyzed using RT-PCR, western blot, and ELISA. Adipogenic differentiation was identified using Oil Red O staining, and the levels of peroxisome proliferator activator gamma (PPARγ) and CCAAT-enhancer-binding proteins (C/EBPs) α and β were determined by western blot. Results The expression of GSK-3β was significantly higher in GO tissues than in control tissues. The addition of CHIR 99021 led to a decrease in the active form of the kinase in which the Y216 residue is phosphorylated. When GO and non-GO fibroblasts were stimulated with IL-1β or TNF-α, IL-6, IL-8, intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-1 (COX-1), and monocyte chemoattractant protein 1 (MCP-1) showed increased production, which was blunted when CHIR 99021 was added. The activation of Akt, PI3K, nuclear factor (NF)-κB, Erk, Jnk, and p38 kinase by IL-1β and TNF-α was diminished with CHIR 99021 in GO cells. A decrease in lipid droplets and expression of PPARγ and c/EBPα and -β was noted in fibroblasts treated with CHIR 99021 during adipocyte differentiation. The inhibition of Wnt and β-catenin in adipogenesis was reversed by CHIR 99021. Conclusions GSK-3β plays a significant role in GO pathogenesis. The inhibition of the kinase attenuated the proinflammatory cytokines production and fibroblast differentiation into adipocytes. GSK-3β may be a potential target for anti-inflammatory and anti-adipogenic treatment of GO.

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Therapeutic Effect of Resveratrol on Oxidative Stress in Graves' Orbitopathy Orbital Fibroblasts

Abstract: Resveratrol reduced ROS levels and inhibited adipogenesis in GO orbital fibroblasts in vitro. This study supports the potential use of resveratrol in GO treatment.

Cited by 26 publications

References 46 publications

Role of Proprotein Convertase Subtilisin/Kexin Type 9 in the Pathogenesis of Graves’ Orbitopathy in Orbital Fibroblasts

Role of Proprotein Convertase Subtilisin/Kexin Type 9 in the Pathogenesis of Graves’ Orbitopathy in Orbital Fibroblasts

Anti-oxidative and anti-adipogenic effects of caffeine in an <i>in vitro</i> model of Graves’ orbitopathy

Glycogen Synthase Kinase-3β Mediates Proinflammatory Cytokine Secretion and Adipogenesis in Orbital Fibroblasts from Patients with Graves’ Orbitopathy

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