title: Antifibrotic effects of crocetin in scleroderma fibroblasts and in bleomycin-induced sclerotic mice.
abstract: OBJECTIVE
  To investigate the antifibrotic effects of crocetin in scleroderma fibroblasts and in sclerotic mice.
  
  
  METHODS
  Skin fibroblasts that were isolated from three systemic scleroderma (SSc) patients and three healthy subjects were treated with crocetin (0.1, 1 or 10 μM). Cell proliferation was measured with an MTT assay. Alpha-smooth muscle actin was detected via an immunohistochemical method. Alpha 1 (I) procollagen (COL1A1), alpha 1 (III) procollagen (COL3A1), matrix metalloproteinase (MMP)-1 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA levels were measured using real-time PCR. SSc mice were established by the subcutaneous injection of bleomycin. Crocetin (50 mg/kg/d) was injected intraperitoneally for 14 days. Dermal thickness and lung fibrosis were assessed with Masson's trichrome staining. Plasma ET-1 was detected with an enzyme-linked immunosorbent assay (ELISA). Skin and lung ET-1 and COL1A1 mRNA levels were measured via real-time PCR.
  
  
  RESULTS
  Crocetin inhibited the proliferation of SSc and normal fibroblasts, an effect that increased with crocetin concentration and incubation time. Crocetin decreased the expression of α-SMA and the levels of mRNA for COL1A1, COL3A1 and matrix metalloproteinase-1, while crocetin increased TIMP-1 mRNA levels in both SSc and normal fibroblasts. Skin and lung fibrosis was induced, and the levels of ET-1 in the plasma, skin and lungs were elevated in bleomycin-injected mice. Crocetin alleviated the thickening of the dermis and lung fibrosis; decreased COL1A1 mRNA levels in the skin and lung; and simultaneously decreased ET-1 concentrations in the plasma and ET-1 mRNA levels in the skin and lungs of the bleomycin-induced sclerotic mice, especially during the early phase (weeks 1-3).
  
  
  CONCLUSIONS
  Crocetin inhibits cell proliferation, differentiation and collagen production in SSc fibroblasts. Crocetin alleviates skin and lung fibrosis in a bleomycin-induced SSc mouse model, in part due to a reduction in ET-1.
authors.affiliation: Fudan University, Zhongshan Hospital, Department of Dermatology, Shanghai, China.
authors.forename: Yinghua
authors.initials: Y
authors.lastname: Song
authors.forename: Lubing
authors.initials: L
authors.lastname: Zhu
authors.forename: Ming
authors.initials: M
authors.lastname: Li
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic; D016588:Anticarcinogenic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001761:Bleomycin; D002338:Carotenoids / Q000494:pharmacology* / Q000627:therapeutic use; D024042:Collagen Type I / Q000097:blood; D000090602:Collagen Type I, alpha 1 Chain; D024061:Collagen Type III / Q000097:blood; D019332:Endothelin-1 / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005355:Fibrosis; D007150:Immunohistochemistry; D008168:Lung / Q000187:drug effects / Q000378:metabolism; D020781:Matrix Metalloproteinase 1 / Q000097:blood; D051379:Mice; D008809:Mice, Inbred C3H; D060888:Real-Time Polymerase Chain Reaction; D012595:Scleroderma, Systemic / Q000139:chemically induced / Q000188:drug therapy* / Q000473:pathology; D012867:Skin / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000097:blood; D014801:Vitamin A / Q000031:analogs & derivatives
chemical_list: D000903:Antibiotics, Antineoplastic; D016588:Anticarcinogenic Agents; C439971:COL3A1 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D024061:Collagen Type III; D019332:Endothelin-1; D019715:Tissue Inhibitor of Metalloproteinase-1; C487773:trans-sodium crocetinate; D001761:Bleomycin; D014801:Vitamin A; D002338:Carotenoids; D020781:Matrix Metalloproteinase 1