主 办:北 京 中 医 药 大 学
ISSN 1006-2157 CN 11-3574/R

JOURNAL OF BEIJIGN UNIVERSITY OF TRADITIONAL CHINE ›› 2018, Vol. 41 ›› Issue (11): 950-958.doi: 10.3969/j.issn.1006-2157.2018.11.011

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Influence of osthole on RAW264.7 cells differentiating to osteoclasts and mechanism study*

Wang Lining1, Ma Yong1,2, Zheng Suyang1, Pan Yalan1,Tu Pengcheng1, Sun Jie1, Guo Yang1#   

  1. 1 Institute of Orthopedics and Traumatology, New Technology on Trauma Repairment and Reconstruction Laboratory, Nanjing University of Chinese Medicine, Nanjing 210023, China;
    2 Department of Orthopaedics, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China
  • Received:2018-05-28 Online:2018-11-30 Published:2018-11-30
  • Supported by:
    National Natural Science Foundation of China (No. 81473692)

Abstract: Objective To investigate the influence of osthole (OST) on RAW264.7 cell lines of mouse monocytes differentiating to osteoclasts and study the related mechanism. Methods Osteoclasts were induced and cultured in vitro by using RAW264.7 cell lines, and then RAW264.7 cell line was divided into negative group, control group and OST group. RAW264.7 cell lines were induced to differentiation by using RANKL, and OST group was intervened with high-dose, mid-dose and low-dose OST (100 μmol/L, 10μmol/L, 1 μmol/L, high-dose, mid-dose and low-dose OST groups) for corresponding time. CCK-8 method was used to screen non-cytotoxic medicinal concentration group, and anti-tartaric acid phosphatase (TRAP) staining method was used to determine number of TRAP positive cells and fusion index. The influence of OST on bone absorptive function was reviewed through detecting area of dissolving pit and fluorescence intensity in bone absorption board. The influence of OST on expressions of nuclear factor of activated T cell (NFAT) and nuclear factor-κB (NF-κB) in RAW264.7 cell lines were observed by using luciferase reporter gene method. The influence of OST on expressions of nuclear factor of activated T cell-1 (NFATc1), CTSK, MMP-9, TRAP, INTE-BETAβ3 and c-Src was detected by using q-PCR. The influence of OST on expressions of p65, IκB, p-IκB and p65 in NF-κB signaling pathway was detected by using Western blotting assay. Results There were 2 medicinal concentration groups (1 μmol/L and 10 μmol/L, low-dose group and mid-dose group) screened by using CCK-8 method. TRAP positive cells and fusion index were significantly lower in all OST groups than those in control group (P<0.05), which was more significant in mid-dose OST group (P<0.05). The related area of bone absorption and fluorescence intensity were significantly inhibited in OST group (P<0.05), which was more significant in mid-dose OST group (P<0.05). OST inhibited significantly the initiations of NFAT and NF-κB (P<0.05) and expressions of osteoclast related genes including NFATc1, and except of Integrin-BETA3 and c-Src, other related genes had statistical difference between low-dose group and mid-dose group (P<0.05). Compared with control group, the protein expression of p-IκB was inhibited significantly (P<0.05), expressions of IκB and p65 were improved (P<0.05), and protein expression of p65 was inhibited (P<0.05) in all OST groups. Conclusion OST can induce down-regulations of NFATc1 and related transcription factors to control differentiation of osteoclasts through inhibiting expression of NF-κB signaling pathway.

Key words: osthole, osteoclasts, nuclear factor of activated T cell-1, osteoporosis, differentiation, nuclear factor-κB signaling pathway

CLC Number: 

  • R285.5