title: Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression.
abstract: OBJECTIVE
  Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro.
  
  
  METHODS
  The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis.
  
  
  RESULTS
  Aconine (0.125, 0.25 μmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, β3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion.
  
  
  CONCLUSIONS
  These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP.
authors.affiliation: School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.|College of Pharmacy, Hainan Medical University, Haikou 571199, China.
authors.forename: Xiang-zhou
authors.initials: XZ
authors.lastname: Zeng
authors.affiliation: School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.
authors.forename: Long-gang
authors.initials: LG
authors.lastname: He
authors.affiliation: Department of Surgery, Guangdong Hospital of Traditional Chinese Medicine, Guangzhou 510120, China.
authors.forename: Song
authors.initials: S
authors.lastname: Wang
authors.affiliation: School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.
authors.forename: Keng
authors.initials: K
authors.lastname: Wang
authors.affiliation: School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.
authors.forename: Yue-yang
authors.initials: YY
authors.lastname: Zhang
authors.affiliation: School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Tao
authors.affiliation: School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.
authors.forename: Xiao-juan
authors.initials: XJ
authors.lastname: Li
authors.affiliation: School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.
authors.forename: Shu-wen
authors.initials: SW
authors.lastname: Liu
mesh_terms: D000157:Aconitine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D001862:Bone Resorption / Q000188:drug therapy / Q000235:genetics / Q000276:immunology; D002454:Cell Differentiation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D016328:NF-kappa B / Q000276:immunology*; D050778:NFATC Transcription Factors / Q000276:immunology*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D053245:RANK Ligand / Q000276:immunology*; D000067996:RAW 264.7 Cells
chemical_list: D000276:Adjuvants, Immunologic; C499636:DC-STAMP protein, mouse; D008565:Membrane Proteins; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D009419:Nerve Tissue Proteins; C494005:Nfatc1 protein, mouse; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse; C047306:aconine; D000157:Aconitine