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 Iα, α-smooth muscle actin with or without TGF-β stimulation, and MMP-2, -7, -9, and tissue inhibitor of metalloproteinase-1 with or without IL-1β stimulation. The gelatinolytic activities of MMP-2 and MMP-9 were measured using gelatin zymography. In tissue cultures, MMP secretion and MMP and collagen Iα 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-β-stimulated fibronectin and collagen Iα, and IL-1β-enhanced MMP-2 and MMP-9. However, without IL-1β stimulation, 10-50 μM 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 μM of quercetin increased tissue MMP-2 mRNA. MMP-9 and collagen Iα 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.
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 Iα, α-smooth muscle actin with or without TGF-β stimulation, and MMP-2, -7, -9, and tissue inhibitor of metalloproteinase-1 with or without IL-1β stimulation. The gelatinolytic activities of MMP-2 and MMP-9 were measured using gelatin zymography. In tissue cultures, MMP secretion and MMP and collagen Iα 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-β-stimulated fibronectin and collagen Iα, and IL-1β-enhanced MMP-2 and MMP-9. However, without IL-1β stimulation, 10-50 μM 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 μM of quercetin increased tissue MMP-2 mRNA. MMP-9 and collagen Iα 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.
Authors: Vardaan Gupta; Christine L Hammond; Elisa Roztocil; Mithra O Gonzalez; Steven E Feldon; Collynn F Woeller Journal: Surv Ophthalmol Date: 2021-09-04 Impact factor: 6.197