title: Antifibrotic effects of quercetin in primary orbital fibroblasts and orbital fat tissue cultures of Graves' orbitopathy.
abstract: OBJECTIVE
  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.affiliation: Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea. yoonjs@yuhs.ac
authors.forename: Jin Sook
authors.initials: JS
authors.lastname: Yoon
authors.forename: Min Kyung
authors.initials: MK
authors.lastname: Chae
authors.forename: Sun Young
authors.initials: SY
authors.lastname: Jang
authors.forename: Sang Yeul
authors.initials: SY
authors.lastname: Lee
authors.forename: Eun Jig
authors.initials: EJ
authors.lastname: Lee
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000273:Adipose Tissue / Q000187:drug effects* / Q000378:metabolism; D000328:Adult; D000975:Antioxidants / Q000494:pharmacology*; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D005355:Fibrosis / Q000517:prevention & control; D049970:Graves Ophthalmopathy / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D006801:Humans; D007770:L-Lactate Dehydrogenase; D008297:Male; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D009915:Orbit / Q000473:pathology*; D009916:Orbital Diseases / Q000473:pathology / Q000517:prevention & control*; D011794:Quercetin / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts; D013844:Thiazoles; D014945:Wound Healing / Q000187:drug effects
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D000975:Antioxidants; D015415:Biomarkers; D024042:Collagen Type I; D005353:Fibronectins; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; D011794:Quercetin; D007770:L-Lactate Dehydrogenase; D020782:Matrix Metalloproteinases; C022616:thiazolyl blue