Journal of Prevention and Treatment for Stomatological Diseases ›› 2018, Vol. 26 ›› Issue (3): 158-165.doi: 10.12016/j.issn.2096-1456.2018.03.004

• Basic Study • Previous Articles     Next Articles

Effect and molecular mechanism of isorhamnetin extracted from Ginkgo biloba on the differentiation of RAW264.7 cells into osteoclasts

Jing LI1, Liang CHENG1, Lühua GUO2, Tong LI1(), Moyang YANG2, Junmei WANG1, Zhe WU1,2()   

  1. 1. Department of Prothodontics, School and Hospital of Stomatology, Jilin University, Changchun 130021, China;
    2. Department of Prothodontics, Stomatology School of Guangzhou Medical University, Guangzhou 510500, China;
  • Received:2017-12-06 Revised:2017-12-24 Online:2018-03-20 Published:2018-08-31

Abstract:

Objective To investigate the effect and potential molecular mechanisms of isorhamnetin (ISO) extracted from Ginkgo biloba on the differentiation of osteoclasts. Methods Osteoclast precursor RAW264.7 cells were induced with RANKL to differentiate into mature osteoclasts. Different concentrations of ISO were added to RAW264.7 cells to determine its effect on osteoclast differentiation. CCK8 was used to evaluate the effect of ISO on cytotoxicity. The impact of ISO on the osteoclast differentiation process was investigated by analyzing tartrate resistance and bone resorption lacuna. Real-time PCR was performed to analyze the levels of differentiation marker genes, including tartrate resistant acid phosphatase (Trap), cathepsin K (Ctsk), and matrix metalloproteinase 9 (MMP-9); differentiation-related transcription factors, including the proto-oncogene protein c-Fos, nuclear factor of activated T-cells cytoplasmic 1(NFATc1); and the levels of downstream NF-κB p65 signaling pathway phosphorylation. Using the above-described method, we verified that ISO exerted an inhibitory effect on osteoclast differentiation and explored related molecular mechanisms. Results Different concentrations of ISO (1-10 μM) had no cytotoxic effects on RAW264.7 cells, inhibited TRAP activity and decreased the number of bone resorption lacuna during osteoclast differentiation. When applied at a concentration of 10 μM, its inhibitory effect was significant. In addition, ISO significantly reduced the expression levels of Trap, Ctsk, MMP-9, c-Fos, NFATc1 and NF-κB p65 mRNA. Conclusion ISO extracted from Ginkgo biloba extract exerted an inhibitory effect on osteoclast differentiation, and the mechanism underlying its activity may involve the inhibition of the classical NF-κB pathway.

Key words: Ginkgo biloba extract, Flavonoids, Isorhamnetin, Osteoclast, Osteoclast differentiation, RANKL, NF-κB;

CLC Number: 

  • R783.4

Figure 1

Different concentrations of RANKL induced different numbers of osteoclasts in RAW264.7 cells cultured for 5 days"

Figure 2

The effect of ISO on the proliferation of RAW264.7 cells"

Figure 3

The results of TRAP staining were affected by differences in the concentrations of ISO × 100"

Figure 4

The relative expression levels of osteoclast differentiation marker genes in RAW264.7 cells were affected by ISO treatment as detected by PCR"

Figure 5

SEM images of bone resorption lacuna × 400"

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