title: Citrus limonoid nomilin inhibits osteoclastogenesis in vitro by suppression of NFATc1 and MAPK signaling pathways.
abstract: BACKGROUND
  Animal experiment studies have revealed a positive association between intake of citrus fruits and bone health. Nomilin, a limonoid present in citrus fruits, is reported to have many biological activities in mammalian systems, but the mechanism of nomilin on bone metabolism regulation is currently unclear.
  
  
  OBJECTIVE
  To reveal the mechanism of nomilin on osteoclastic differentiation of mouse primary bone marrow-derived macrophages (BMMs) and the mouse RAW 264.7 macrophage cell line into osteoclasts.
  
  
  METHODS
  Controlled laboratory study. Effects of nomilin on osteoclastic differentiation were studied in in vitro cell cultures.
  
  
  METHODS
  Cell viability of RAW 264.7 cells and BMMs was measured with the Cell Counting Kit. TRAP-positive multinucleated cells were counted as osteoclast cell numbers. The number and area of resorption pits were measured as bone-resorbing activity. Osteoclast-specific genes expression was evaluated by quantitative real-time PCR; and proteins expression was evaluated by western blot.
  
  
  RESULTS
  Nomilin significantly decreased TRAP-positive multinucleated cell numbers compared with the control, and exhibited no cytotoxicity. Nomilin decreased bone resorption activity. Nomilin downregulated osteoclast-specific genes, NFATc1 and TRAP mRNA levels. Furthermore, nomilin suppressed MAPK signaling pathways.
  
  
  CONCLUSIONS
  This study demonstrates clearly that nomilin has inhibitory effects on osteoclastic differentiation in vitro. These findings indicate that nomilin-containing herbal preparations have potential utility for the prevention of bone metabolic diseases.
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan. Electronic address: kimira@josai.ac.jp.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Kimira
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Taniuchi
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors.forename: Sachie
authors.initials: S
authors.lastname: Nakatani
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors.forename: Yuusuke
authors.initials: Y
authors.lastname: Sekiguchi
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors.forename: Hyoun Ju
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Shimizu
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Ebata
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Wada
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors.forename: Akiyo
authors.initials: A
authors.lastname: Matsumoto
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mano
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D001584:Benzoxepins / Q000494:pharmacology*; D001862:Bone Resorption; D002454:Cell Differentiation / Q000187:drug effects; D002957:Citrus / Q000737:chemistry*; D007527:Isoenzymes / Q000378:metabolism; D036701:Limonins / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008264:Macrophages / Q000187:drug effects; D008297:Male; D051379:Mice; D050778:NFATC Transcription Factors / Q000378:metabolism*; D010010:Osteoclasts / Q000187:drug effects*; D000067996:RAW 264.7 Cells / Q000187:drug effects; D000071681:Tartrate-Resistant Acid Phosphatase
chemical_list: D001584:Benzoxepins; D007527:Isoenzymes; D036701:Limonins; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; C059405:nomilin; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase
keywords: Limonoid; Nomilin; Osteoclastic differentiation