title: Xanthotoxin prevents bone loss in ovariectomized mice through the inhibition of RANKL-induced osteoclastogenesis.
abstract: Xanthotoxin (XAT) is extracted from the seeds of Ammi majus. Here, we reported that XAT has an inhibitory effect on osteoclastogenesis in vitro through the suppression of both receptor activator of nuclear factor-κB ligand (RANKL)-induced ROS generation and Ca(2+) oscillations. In vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, and restores bone strength in ovariectomized mice.
  
  
  
  Excessive osteoclast formation and the resultant increase in bone resorption activity are key pathogenic factors of osteoporosis. In the present study, we have investigated the effects of XAT, a natural furanocoumarin, on the RANKL-mediated osteoclastogenesis in vitro and on ovariectomy-mediated bone loss in vivo.
  
  
  
  Cytotoxicity of XAT was evaluated using bone marrow macrophages (BMMs). Osteoclast differentiation, formation, and fusion were assessed using the tartrate-resistant acid phosphatase (TRAP) stain, the actin cytoskeleton and focal adhesion (FAK) stain, and the fusion assay, respectively. Osteoclastic bone resorption was evaluated using the pit formation assay. Reactive oxygen species (ROS) generation and removal were evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA). Ca(2+) oscillations and their downstream signaling targets were then detected. The ovariectomized (OVX) mouse model was adopted for our in vivo studies.
  
  
  
  In vitro assays revealed that XAT inhibited the differentiation, formation, fusion, and bone resorption activity of osteoclasts. The inhibitory effect of XAT on osteoclastogenesis was associated with decreased intracellular ROS generation. XAT treatment also suppressed RANKL-induced Ca(2+) oscillations and the activation of the resultant downstream calcium-CaMKK/PYK2 signaling. Through these two mechanisms, XAT downregulated the key osteoclastogenic factors nuclear factor of activated T cells c1 (NFATc1) and c-FOS. Our in vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, rescues bone microarchitecture, and restores bone strength in OVX mice.
  
  
  
  Our findings indicate that XAT is protective against ovariectomy-mediated bone loss through the inhibition of RANKL-mediated osteoclastogenesis. Therefore, XAT may be considered to be a new therapeutic candidate for treating osteoporosis.
authors.affiliation: Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing, 400038, China.|Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China.
authors.forename: C
authors.initials: C
authors.lastname: Dou
authors.affiliation: Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing, 400038, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing, 400038, China.
authors.forename: N
authors.initials: N
authors.lastname: Ding
authors.affiliation: Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing, 400038, China.
authors.forename: N
authors.initials: N
authors.lastname: Li
authors.affiliation: Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing, 400038, China.
authors.forename: H
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing, 400038, China.
authors.forename: C
authors.initials: C
authors.lastname: Zhao
authors.affiliation: Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing, 400038, China.
authors.forename: F
authors.initials: F
authors.lastname: Kang
authors.affiliation: Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing, 400038, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Cao
authors.affiliation: Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing, 400038, China.
authors.forename: H
authors.initials: H
authors.lastname: Quan
authors.affiliation: Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China.
authors.forename: F
authors.initials: F
authors.lastname: Luo
authors.affiliation: Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China.
authors.forename: J
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing, 400038, China. dongshiwu@tmmu.edu.cn.
authors.forename: S
authors.initials: S
authors.lastname: Dong
mesh_terms: D000818:Animals; D001862:Bone Resorption; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D005260:Female; D008264:Macrophages / Q000187:drug effects; D008730:Methoxsalen / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010010:Osteoclasts / Q000187:drug effects*; D010012:Osteogenesis / Q000187:drug effects*; D010052:Ovariectomy; D053245:RANK Ligand / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D053245:RANK Ligand; D017382:Reactive Oxygen Species; C505689:Tnfsf11 protein, mouse; D002118:Calcium; D008730:Methoxsalen
keywords: Bone loss; Calcium oscillation; Osteoclasts; ROS; Xanthotoxin
references.citation: Ann Biomed Eng. 2014 Jun;42(6):1250-60
references.pmid: 24710796
references.citation: J Clin Invest. 2014 Nov;124(11):5057-73
references.pmid: 25329696
references.citation: Clin Chem Lab Med. 2008;46(11):1550-5
references.pmid: 18847368
references.citation: Asia Pac J Clin Nutr. 2007;16 Suppl 1:17-21
references.pmid: 17392070
references.citation: Zhejiang Da Xue Xue Bao Yi Xue Ban. 2009 Jul;38(4):348-51
references.pmid: 19693970
references.citation: Biochim Biophys Acta. 2014 Nov;1839(11):1084-96
references.pmid: 25123438
references.citation: Drugs. 1998 Oct;56(4):667-90
references.pmid: 9806110
references.citation: J Gen Physiol. 1994 Mar;103(3):365-88
references.pmid: 8195779
references.citation: J Photochem Photobiol B. 2006 Oct 2;85(1):39-48
references.pmid: 16735125
references.citation: Nat Commun. 2014 Apr 30;5:3773
references.pmid: 24781012
references.citation: Planta Med. 1995 Oct;61(5):481-2
references.pmid: 17238101
references.citation: Nat Med. 2006 Dec;12(12):1410-6
references.pmid: 17128269
references.citation: Adv Exp Med Biol. 2012;740:917-32
references.pmid: 22453976
references.citation: Lancet. 2002 May 18;359(9319):1761-7
references.pmid: 12049882
references.citation: Biochim Biophys Acta. 2011 May;1813(5):979-83
references.pmid: 21075150
references.citation: J Clin Invest. 2013 Nov;123(11):4731-8
references.pmid: 24216508
references.citation: Cell. 1998 Apr 17;93(2):165-76
references.pmid: 9568710
references.citation: J Nat Med. 2014 Jan;68(1):83-94
references.pmid: 23649674
references.citation: Photodermatol Photoimmunol Photomed. 2011 Dec;27(6):294-6
references.pmid: 22092732
references.citation: Nat Med. 2009 Mar;15(3):259-66
references.pmid: 19252502
references.citation: Exp Cell Res. 2004 Dec 10;301(2):119-27
references.pmid: 15530848
references.citation: J Clin Invest. 2003 Sep;112(6):915-23
references.pmid: 12975476
references.citation: Bone. 2006 Feb;38(2 Suppl 1):S4-9
references.pmid: 16455317
references.citation: J Bone Miner Res. 1992 Sep;7(9):1005-10
references.pmid: 1414493
references.citation: Biochem Biophys Res Commun. 2012 Feb 10;418(2):247-53
references.pmid: 22252298
references.citation: Ann N Y Acad Sci. 1991 Dec 30;636:217-26
references.pmid: 1793211
references.citation: Hematol Oncol Clin North Am. 1995 Oct;9(5):1117-26
references.pmid: 8522488
references.citation: Lancet. 2011 Apr 9;377(9773):1276-87
references.pmid: 21450337
references.citation: Life Sci. 2000 Jan 14;66(8):709-23
references.pmid: 10680579
references.citation: J Cell Physiol. 2016 Mar;231(3):558-67
references.pmid: 25545964
references.citation: Osteoporos Int. 2009 Oct;20(10):1651-62
references.pmid: 19415374
references.citation: J Immunol. 2002 Dec 1;169(11):6427-34
references.pmid: 12444151
references.citation: FASEB J. 1997 Feb;11(2):118-24
references.pmid: 9039953
references.citation: Endocr Rev. 2010 Jun;31(3):266-300
references.pmid: 20051526
references.citation: Blood. 2005 Aug 1;106(3):852-9
references.pmid: 15817678
references.citation: Biofizika. 1982 Nov-Dec;27(6):995-9
references.pmid: 6760905
references.citation: J Clin Invest. 1990 Mar;85(3):632-9
references.pmid: 2312718