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Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops.

Li J, Zeng L, Xie J, Yue Z, Deng H, Ma X, Zheng C, Wu X, Luo J, Liu M - Sci Rep (2015)

Bottom Line: In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems.At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis.As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Fengxian District Central Hospital and East China Normal University Joint Center for Translational Medicine, Shanghai Key laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.

ABSTRACT
Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis.

No MeSH data available.


Related in: MedlinePlus

XN suppresses human and mouse osteoclast differentiation.(A) The effect of xanthohumol (XN) on mouse BMMs differentiation. BMMs were incubated with M-CSF (20 ng/ml) and RANKL (30 ng/ml), followed by addition of different concentrations of XN for 6 days. The cells were stained for TRAP assay and photographed (×40; left). The numbers of TRAP positive multinucleated (>5 nuclei) osteoclasts were counted (right). (B) The effect of XN on RAW264.7 cell differentiation. RAW264.7 cells were treated with RANKL (30 ng/ml) and different doses of XN for 3 days. The cells were stained for TRAP assay and photographed (×40; left) and the numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). (C) The effect of XN on human PBMC differentiation. PBMCs (5 × 105 cells) were stimulated with hRANKL (50 ng/mL) and hCSF1 (20 ng/mL) for 8–9 days. The cells were stained for TRAP assay and photographed (×40; left) and the numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). (D) XN inhibits RANKL-induced osteoclast differentiation at the early stage. Osteoclast precursor BMMs were cultured with M-CSF and RANKL for differentiation into mature osteoclasts in 6 days. XN (5 μM) were added at indicated time (day). The cells were fixed and stained for TRAP activity. (E) The effect of XN on cell viability in BMMs and RAW264.7 cells. BMMs or RAW264.7 cells were treated with different concentrations of XN for 5 days, and the cell viability was measured by SRB assay. (F) The effect of XN on cell viability in human PBMC cells. Human PBMC cells were incubated with hCSF1 (20 ng/mL) and different concentrations of XN for 4 days. The cell viability was measured by SRB assay. Column, means of experiments performed in triplicate; bar, SD. *p < 0.05, **p < 0.01, ***p < 0.001. N.S., no significant.
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f1: XN suppresses human and mouse osteoclast differentiation.(A) The effect of xanthohumol (XN) on mouse BMMs differentiation. BMMs were incubated with M-CSF (20 ng/ml) and RANKL (30 ng/ml), followed by addition of different concentrations of XN for 6 days. The cells were stained for TRAP assay and photographed (×40; left). The numbers of TRAP positive multinucleated (>5 nuclei) osteoclasts were counted (right). (B) The effect of XN on RAW264.7 cell differentiation. RAW264.7 cells were treated with RANKL (30 ng/ml) and different doses of XN for 3 days. The cells were stained for TRAP assay and photographed (×40; left) and the numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). (C) The effect of XN on human PBMC differentiation. PBMCs (5 × 105 cells) were stimulated with hRANKL (50 ng/mL) and hCSF1 (20 ng/mL) for 8–9 days. The cells were stained for TRAP assay and photographed (×40; left) and the numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). (D) XN inhibits RANKL-induced osteoclast differentiation at the early stage. Osteoclast precursor BMMs were cultured with M-CSF and RANKL for differentiation into mature osteoclasts in 6 days. XN (5 μM) were added at indicated time (day). The cells were fixed and stained for TRAP activity. (E) The effect of XN on cell viability in BMMs and RAW264.7 cells. BMMs or RAW264.7 cells were treated with different concentrations of XN for 5 days, and the cell viability was measured by SRB assay. (F) The effect of XN on cell viability in human PBMC cells. Human PBMC cells were incubated with hCSF1 (20 ng/mL) and different concentrations of XN for 4 days. The cell viability was measured by SRB assay. Column, means of experiments performed in triplicate; bar, SD. *p < 0.05, **p < 0.01, ***p < 0.001. N.S., no significant.

Mentions: In order to determine the effect of XN on osteoclastogenesis, we employed three standard in vitro osteoclast differentiation models, mouse BMMs with RANKL and M-CSF stimulation model, RAW264.7 cells with RANKL stimulation model, and human PBMC cells with RANKL and M-CSF stimulation model. As shown in Fig. 1A–C, TRAP positive multinucleated osteoclasts were formed in response to RANKL treatment. However, administration of XN reduced osteoclast differentiation in a dose-dependent manner in all of the three cell models. The half maximal inhibitory concentration (IC50) value of mouse osteoclast is about 1 μM, while the IC50 value of human is between 0.25 and 0.5 μM.


Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops.

Li J, Zeng L, Xie J, Yue Z, Deng H, Ma X, Zheng C, Wu X, Luo J, Liu M - Sci Rep (2015)

XN suppresses human and mouse osteoclast differentiation.(A) The effect of xanthohumol (XN) on mouse BMMs differentiation. BMMs were incubated with M-CSF (20 ng/ml) and RANKL (30 ng/ml), followed by addition of different concentrations of XN for 6 days. The cells were stained for TRAP assay and photographed (×40; left). The numbers of TRAP positive multinucleated (>5 nuclei) osteoclasts were counted (right). (B) The effect of XN on RAW264.7 cell differentiation. RAW264.7 cells were treated with RANKL (30 ng/ml) and different doses of XN for 3 days. The cells were stained for TRAP assay and photographed (×40; left) and the numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). (C) The effect of XN on human PBMC differentiation. PBMCs (5 × 105 cells) were stimulated with hRANKL (50 ng/mL) and hCSF1 (20 ng/mL) for 8–9 days. The cells were stained for TRAP assay and photographed (×40; left) and the numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). (D) XN inhibits RANKL-induced osteoclast differentiation at the early stage. Osteoclast precursor BMMs were cultured with M-CSF and RANKL for differentiation into mature osteoclasts in 6 days. XN (5 μM) were added at indicated time (day). The cells were fixed and stained for TRAP activity. (E) The effect of XN on cell viability in BMMs and RAW264.7 cells. BMMs or RAW264.7 cells were treated with different concentrations of XN for 5 days, and the cell viability was measured by SRB assay. (F) The effect of XN on cell viability in human PBMC cells. Human PBMC cells were incubated with hCSF1 (20 ng/mL) and different concentrations of XN for 4 days. The cell viability was measured by SRB assay. Column, means of experiments performed in triplicate; bar, SD. *p < 0.05, **p < 0.01, ***p < 0.001. N.S., no significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664947&req=5

f1: XN suppresses human and mouse osteoclast differentiation.(A) The effect of xanthohumol (XN) on mouse BMMs differentiation. BMMs were incubated with M-CSF (20 ng/ml) and RANKL (30 ng/ml), followed by addition of different concentrations of XN for 6 days. The cells were stained for TRAP assay and photographed (×40; left). The numbers of TRAP positive multinucleated (>5 nuclei) osteoclasts were counted (right). (B) The effect of XN on RAW264.7 cell differentiation. RAW264.7 cells were treated with RANKL (30 ng/ml) and different doses of XN for 3 days. The cells were stained for TRAP assay and photographed (×40; left) and the numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). (C) The effect of XN on human PBMC differentiation. PBMCs (5 × 105 cells) were stimulated with hRANKL (50 ng/mL) and hCSF1 (20 ng/mL) for 8–9 days. The cells were stained for TRAP assay and photographed (×40; left) and the numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). (D) XN inhibits RANKL-induced osteoclast differentiation at the early stage. Osteoclast precursor BMMs were cultured with M-CSF and RANKL for differentiation into mature osteoclasts in 6 days. XN (5 μM) were added at indicated time (day). The cells were fixed and stained for TRAP activity. (E) The effect of XN on cell viability in BMMs and RAW264.7 cells. BMMs or RAW264.7 cells were treated with different concentrations of XN for 5 days, and the cell viability was measured by SRB assay. (F) The effect of XN on cell viability in human PBMC cells. Human PBMC cells were incubated with hCSF1 (20 ng/mL) and different concentrations of XN for 4 days. The cell viability was measured by SRB assay. Column, means of experiments performed in triplicate; bar, SD. *p < 0.05, **p < 0.01, ***p < 0.001. N.S., no significant.
Mentions: In order to determine the effect of XN on osteoclastogenesis, we employed three standard in vitro osteoclast differentiation models, mouse BMMs with RANKL and M-CSF stimulation model, RAW264.7 cells with RANKL stimulation model, and human PBMC cells with RANKL and M-CSF stimulation model. As shown in Fig. 1A–C, TRAP positive multinucleated osteoclasts were formed in response to RANKL treatment. However, administration of XN reduced osteoclast differentiation in a dose-dependent manner in all of the three cell models. The half maximal inhibitory concentration (IC50) value of mouse osteoclast is about 1 μM, while the IC50 value of human is between 0.25 and 0.5 μM.

Bottom Line: In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems.At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis.As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Fengxian District Central Hospital and East China Normal University Joint Center for Translational Medicine, Shanghai Key laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.

ABSTRACT
Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis.

No MeSH data available.


Related in: MedlinePlus