Antifibrotic Effects of Quercetin in Primary Orbital Fibroblasts and Orbital Fat Tissue Cultures of Graves' Orbitopathy

Abstract

PURPOSE. We investigated the effects of quercetin on fibrotic markers and matrix metalloproteinases (MMPs) in primary cells and whole orbital tissues from Graves' orbitopathy (GO). METHODS. Orbital fat tissues were harvested from GO for primary cell and tissue cultures during orbital fat decompression. To determine noncytotoxic dose and time of quercetin treatment, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and LDH release assay were performed. The effects of quercetin on fibrosis were evaluated according to a scratch wound closure assay, and Western blotting for expression of fibronectin, collagen I alpha, alpha-smooth muscle actin with or without TGF-beta stimulation, and MMP-2, -7, -9, and tissue inhibitor of metalloproteinase-1 with or without IL-1 beta stimulation. The gelatinolytic activities of MMP-2 and MMP-9 were measured using gelatin zymography. In tissue cultures, MMP secretion and MMP and collagen I alpha mRNA levels were determined by enzyme-linked immunosorbent assays and reverse transcription-polymerase chain reaction (RT-PCR), respectively. RESULTS. Quercetin significantly inhibited cell migration at nontoxic concentrations. In primary cells, quercetin dose-dependently downregulated expression of TGF-beta-stimulated fibronectin and collagen I alpha, and IL-1 beta-enhanced MMP-2 and MMP-9. However, without IL-1 beta stimulation, 10-50 mu M of quercetin increased MMP-2 expression and activity, but dose-dependently suppressed MMP-9 expression and activity. In tissue cultures, quercetin dose-dependently inhibited MMP-2 and -9 activity and secretion, but 30 and 50 mu M of quercetin increased tissue MMP-2 mRNA. MMP-9 and collagen I alpha mRNA levels were dose-dependently suppressed. CONCLUSIONS. Quercetin inhibited fibrotic markers and affected MMP-2 and MMP-9 activities in primary cell and orbital fat tissue cultures from GO at nontoxic concentrations. Our results support the potential use of quercetin for active inflammation and treatment or prevention of chronic fibrosis in GO. (Invest Ophthalmol Vis Sci. 2012;53:5921-5929) DOI: 10.1167/iovs.12-9646