Quercetin inhibits IL-1β-induced inflammation, hyaluronan production and adipogenesis in orbital fibroblasts from Graves' orbitopathy

Abstract

Management of Graves' orbitopathy (GO) is challenging, as no reliable, specific, and safe medical therapeutic agents have yet been developed. We investigated the effect of quercetin in primary cultured orbital fibroblasts from GO, targeting pathways of inflammation, aberrant accumulation of extracellular matrix macromolecules, and adipose tissue expansion. Quercetin significantly attenuated intercellular adhesion molecule-1 (ICAM-1), interleukin (IL) -6, IL-8, and cyclooxygenase (COX) -2 mRNA expression, and inhibited IL-1β-induced increases in ICAM-1, IL-6, and IL-8 mRNA. Increased hyaluronan production induced by IL-1β or tumor necrosis factor-α was suppressed by quercetin in a dose- and time-dependent manner. Treatment with noncytotoxic doses of quercetin inhibited accumulation of intracytoplasmic lipid droplets and resulted in a dose-dependent decrease in expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein (C/EBP) α, and C/EBPβ proteins. In conclusion, inhibition of inflammation, hyaluronan production, and adipogenesis by the natural plant product quercetin in vitro provides the basis for further study of its potential use in the treatment of GO.

Figure 1. Effect of quercetin on cell viability and apoptosis in preadipocyte orbital fibroblasts and differentiating orbital fibroblasts.

(A) Orbital fibroblasts (1×10⁵) of normal and Graves' orbitopathy (GO) patients were seeded into 24-well culture plates and treated with different concentrations of quercetin (10, 30, 50, or 100 µM) for 24 h. After treatment, assays with 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) were performed to test for viability. (B) An annexin V/FITC kit was used to detect phosphatidylserine externalization, as an index of apoptosis. Percentage of stained cells with annexin V was analyzed by flow cytometry. (C) Orbital fibroblasts (1×10⁵) of GO patients were seeded into 24-well culture plates and treated with different concentrations of quercetin (10, 50, 100, or 200 µM) for 3 days in adipogenic medium containing adipogenesis inducers and rosiglitazone (10 µM). After treatment, MTT assays were performed. Results are expressed as percentage of untreated control values presented as mean ± standard deviation (SD). Assays were performed at least three times in triplicate; data from a representative experiment are shown, expressed as the differences between treated and untreated cells.

Figure 2. Effects of quercetin on ICAM-1, IL-6, IL-8, and COX-2 mRNA expression in Graves' orbitopathy (GO) orbital fibroblasts.

(A) Orbital fibroblasts (5×10⁵) from GO patients pretreated with 0, 50, or 100 µM quercetin for 24 h were used to analyze for ICAM-1, IL-6, IL-8, and COX-2 mRNA expression by RT-PCR. (B) Cells pretreated as in (A) were then stimulated with IL-1β (10 ng/ml) for 16 h, and were then used for RT-PCR analyses. (C) RT-PCR analysis of ICAM-1, IL-6, IL-8, and COX-2 mRNA expression, with values determined by densitometry and normalized to GAPDH. Cells had been pretreated with 100 µM quercetin for 6, 9, or 24 h, then stimulated with IL-1β (10 ng/ml) for 16 h. Data in each column above represent the mean relative density ratio ± SD of three experiments, and representative gel images are shown below the graphs. Differences between treated and untreated cells (*P<0.05, **P<0.001) are indicated.

Figure 3. The effect of quercetin on hyaluronan production induced by IL-1β or TNF-α in orbital fibroblasts.

(A) Hyaluronan in media of confluent orbital fibroblast cultures from GO (n = 3) and normal (n = 3) individuals pretreated with 0, 10, 50 or 100 µM quercetin for 24 h before IL-1β stimulation (10 ng/ml, 16 h). (B) The effect of quercetin on hyaluronan production in GO orbital fibroblasts (n = 3) stimulated with IL-1β (10 ng/ml, 16 h) or TNF-α (10 ng/ml, 16 h). Triplicate measurements were averaged, and the data are expressed as mean values ± SD. *P<0.05 vs. cells stimulated with IL-1β or TNF-α alone.

Figure 4. Effect of quercetin on NF-κB activation in GO orbital fibroblasts.

(A) Cells were pretreated with quercetin for 24 h prior to IL-1β (10 ng/ml) stimulation for 16 h, and p65 NF-κB translocation was assayed by western blot analysis. (B) Results of assays measuring NF-κB activity with a NF-κB-dependent luciferase reporter construct in cells treated with quercetin (0, 50 or 100 µM, 24 h) prior to stimulation with IL-1β or TNF-α (10 ng/ml) for 16 h. (C) RT-PCR analysis of ICAM-1, IL-6, IL-8, and COX-2 expression in cells pretreated with NF-κB inhibitor SC-514 (100 µM) for 1 h and then stimulated with IL-1β (10 ng/ml). PCR bands measured by densitometry and normalized to GAPDH. *P<0.05 vs. cells stimulated with IL-1β or TNF-α alone.

Figure 5. Effect of quercetin on adipogenesis in GO orbital fibroblasts.

(A–B) Quercetin (50 or 100 µM) treatment for the first 3 days after initiation of 10-day adipogenesis in adipogenic media containing (A) 10 µM rosiglitazone, or (B) combined 10 µM rosiglitazone and 10 ng/ml IL-1β. Cells were stained with Oil Red O and examined grossly and microscopically (×40; inset ×400). (C) Cell-bound Oil Red O was solubilized and optical density (OD) read at 490 nm to obtain a quantitative assessment of adipogenesis. The experiments were performed in triplicate with cells from three different donors, and data in the column are the mean relative density ratios ± SD of three experiments. *P<0.001 vs. untreated control differentiated cells.

Figure 6. Effect of quercetin on the expression of adipogenic transcriptional regulators in differentiated orbital fibroblasts from GO patients.

(A) Quercetin (50 or 100 µM) treatment for the first 3 days after initiation of 10-day adipogenesis in adipogenic media containing 10 µM rosiglitazone, or combined 10 µM rosiglitazone and 10 ng/ml IL-1β. After 10 days, cell lysates were subjected to western blot analysis of PPARγ, C/EBPα, and C/EBPβ protein expression. The experiments were performed in triplicate with cells from three different donors. (B–D) Quantification by densitometry, normalized to the β-actin level in the same sample, is shown for PPARγ (B), C/EBPα (C), and C/EBPβ (D). The data in the column are the mean relative density ratios ± SD of three experiments. *P<0.05, **P<0.001 vs. untreated control differentiated cells.