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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 RANKL-induced NF-κB signaling pathway, but has little effect on MAPK/AP-1 signaling.(A) The effect of XN on RANKL-induced IκBα degradation and p65 phorsphorylation. RAW264.7 cells were pretreated with XN (5 μM) for 3 hours, and then stimulated with RANKL (30 ng/ml) for indicated time. The degradation of IκBα and the phosphorylation of p65 were tested by Western blot analysis (left). The Western blot were performed in triplicate. The IκBα protein level (with β-actin for normalization) were quantified by Quantity One software (right). (B) The effect of XN on RANKL-induced p65 nuclear translocation. RAW264.7 cells were pretreated with different doses of XN for 3 hours, and then stimulated with RANKL for 20 minutes. Cell Nuclear Extracts (NE) and Cytoplasmic Extract (CE) were collected and subjected to Western blot analysis with the indicated antibodies. (C) The effect of XN on RANKL-induced activity of NF-κB. RAW264.7 cells were co-transfected with NF-κB-luciferase reporter gene and Renilla gene. After 48 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured. (D) NF-κB (p65) prevents the inhibitory effect of XN in RANKL-induced osteoclast differentiation. RAW264.7 cells were transfected with p65 or control plasmids, and then incubated with or without XN (5 μM) in the presence of RANKL (30 ng/ml). After 4 days, the cells were stained for TRAP activity and the numbers of osteoclasts were counted. (E) XN has little effect on RANKL-induced phosphorylation of MAPKs. BMMs were cultured in the presence of XN (5 μM) for 4 hours, and then RANKL was stimulated at the indicated time points. Cell lysates were extracted for Western blot analysis with indicated antibodies. (F) XN has little effect on the activity of AP-1 induced by RANKL. RAW264.7 cells were co-transfected with AP-1-luciferase reporter gene and Renilla gene. After 48 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured. Column, means of experiments conducted in triplicate; bar, SD. *p < 0.05, **p < 0.01, ***p < 0.001.
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f6: XN suppresses RANKL-induced NF-κB signaling pathway, but has little effect on MAPK/AP-1 signaling.(A) The effect of XN on RANKL-induced IκBα degradation and p65 phorsphorylation. RAW264.7 cells were pretreated with XN (5 μM) for 3 hours, and then stimulated with RANKL (30 ng/ml) for indicated time. The degradation of IκBα and the phosphorylation of p65 were tested by Western blot analysis (left). The Western blot were performed in triplicate. The IκBα protein level (with β-actin for normalization) were quantified by Quantity One software (right). (B) The effect of XN on RANKL-induced p65 nuclear translocation. RAW264.7 cells were pretreated with different doses of XN for 3 hours, and then stimulated with RANKL for 20 minutes. Cell Nuclear Extracts (NE) and Cytoplasmic Extract (CE) were collected and subjected to Western blot analysis with the indicated antibodies. (C) The effect of XN on RANKL-induced activity of NF-κB. RAW264.7 cells were co-transfected with NF-κB-luciferase reporter gene and Renilla gene. After 48 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured. (D) NF-κB (p65) prevents the inhibitory effect of XN in RANKL-induced osteoclast differentiation. RAW264.7 cells were transfected with p65 or control plasmids, and then incubated with or without XN (5 μM) in the presence of RANKL (30 ng/ml). After 4 days, the cells were stained for TRAP activity and the numbers of osteoclasts were counted. (E) XN has little effect on RANKL-induced phosphorylation of MAPKs. BMMs were cultured in the presence of XN (5 μM) for 4 hours, and then RANKL was stimulated at the indicated time points. Cell lysates were extracted for Western blot analysis with indicated antibodies. (F) XN has little effect on the activity of AP-1 induced by RANKL. RAW264.7 cells were co-transfected with AP-1-luciferase reporter gene and Renilla gene. After 48 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured. Column, means of experiments conducted in triplicate; bar, SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Mentions: RANKL-induced NF-κB signaling pathway is another essential early molecular event during osteoclastogenesis2425. Therefore, we next examined whether XN had any inhibitory effect on RANKL-induced NF-κB signaling pathway. Using Western blot assay, we verified that XN inhibited the degradation of IκBα and the phosphorylation of NF-κB/p65 induced by RANKL (Fig. 6A). Furthermore, XN suppressed RANKL-stimulated nuclear translocation of NF-κB/p65 in a dose-dependent manner (Fig. 6B). Similar results were obtained by the luciferase reporter gene assay that XN concentration-dependently suppressed RANKL-induced NF-κB activity (Fig. 6C). As expected, NF-κB/p65 prevents the inhibitory effect of XN in RANKL-induced osteoclastogenesis (Fig. 6D). Together, all of the data indicate a role of XN in the modulation of NF-κB signaling pathways.


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 RANKL-induced NF-κB signaling pathway, but has little effect on MAPK/AP-1 signaling.(A) The effect of XN on RANKL-induced IκBα degradation and p65 phorsphorylation. RAW264.7 cells were pretreated with XN (5 μM) for 3 hours, and then stimulated with RANKL (30 ng/ml) for indicated time. The degradation of IκBα and the phosphorylation of p65 were tested by Western blot analysis (left). The Western blot were performed in triplicate. The IκBα protein level (with β-actin for normalization) were quantified by Quantity One software (right). (B) The effect of XN on RANKL-induced p65 nuclear translocation. RAW264.7 cells were pretreated with different doses of XN for 3 hours, and then stimulated with RANKL for 20 minutes. Cell Nuclear Extracts (NE) and Cytoplasmic Extract (CE) were collected and subjected to Western blot analysis with the indicated antibodies. (C) The effect of XN on RANKL-induced activity of NF-κB. RAW264.7 cells were co-transfected with NF-κB-luciferase reporter gene and Renilla gene. After 48 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured. (D) NF-κB (p65) prevents the inhibitory effect of XN in RANKL-induced osteoclast differentiation. RAW264.7 cells were transfected with p65 or control plasmids, and then incubated with or without XN (5 μM) in the presence of RANKL (30 ng/ml). After 4 days, the cells were stained for TRAP activity and the numbers of osteoclasts were counted. (E) XN has little effect on RANKL-induced phosphorylation of MAPKs. BMMs were cultured in the presence of XN (5 μM) for 4 hours, and then RANKL was stimulated at the indicated time points. Cell lysates were extracted for Western blot analysis with indicated antibodies. (F) XN has little effect on the activity of AP-1 induced by RANKL. RAW264.7 cells were co-transfected with AP-1-luciferase reporter gene and Renilla gene. After 48 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured. Column, means of experiments conducted in triplicate; bar, SD. *p < 0.05, **p < 0.01, ***p < 0.001.
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Related In: Results  -  Collection

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Show All Figures
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f6: XN suppresses RANKL-induced NF-κB signaling pathway, but has little effect on MAPK/AP-1 signaling.(A) The effect of XN on RANKL-induced IκBα degradation and p65 phorsphorylation. RAW264.7 cells were pretreated with XN (5 μM) for 3 hours, and then stimulated with RANKL (30 ng/ml) for indicated time. The degradation of IκBα and the phosphorylation of p65 were tested by Western blot analysis (left). The Western blot were performed in triplicate. The IκBα protein level (with β-actin for normalization) were quantified by Quantity One software (right). (B) The effect of XN on RANKL-induced p65 nuclear translocation. RAW264.7 cells were pretreated with different doses of XN for 3 hours, and then stimulated with RANKL for 20 minutes. Cell Nuclear Extracts (NE) and Cytoplasmic Extract (CE) were collected and subjected to Western blot analysis with the indicated antibodies. (C) The effect of XN on RANKL-induced activity of NF-κB. RAW264.7 cells were co-transfected with NF-κB-luciferase reporter gene and Renilla gene. After 48 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured. (D) NF-κB (p65) prevents the inhibitory effect of XN in RANKL-induced osteoclast differentiation. RAW264.7 cells were transfected with p65 or control plasmids, and then incubated with or without XN (5 μM) in the presence of RANKL (30 ng/ml). After 4 days, the cells were stained for TRAP activity and the numbers of osteoclasts were counted. (E) XN has little effect on RANKL-induced phosphorylation of MAPKs. BMMs were cultured in the presence of XN (5 μM) for 4 hours, and then RANKL was stimulated at the indicated time points. Cell lysates were extracted for Western blot analysis with indicated antibodies. (F) XN has little effect on the activity of AP-1 induced by RANKL. RAW264.7 cells were co-transfected with AP-1-luciferase reporter gene and Renilla gene. After 48 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured. Column, means of experiments conducted in triplicate; bar, SD. *p < 0.05, **p < 0.01, ***p < 0.001.
Mentions: RANKL-induced NF-κB signaling pathway is another essential early molecular event during osteoclastogenesis2425. Therefore, we next examined whether XN had any inhibitory effect on RANKL-induced NF-κB signaling pathway. Using Western blot assay, we verified that XN inhibited the degradation of IκBα and the phosphorylation of NF-κB/p65 induced by RANKL (Fig. 6A). Furthermore, XN suppressed RANKL-stimulated nuclear translocation of NF-κB/p65 in a dose-dependent manner (Fig. 6B). Similar results were obtained by the luciferase reporter gene assay that XN concentration-dependently suppressed RANKL-induced NF-κB activity (Fig. 6C). As expected, NF-κB/p65 prevents the inhibitory effect of XN in RANKL-induced osteoclastogenesis (Fig. 6D). Together, all of the data indicate a role of XN in the modulation of NF-κB signaling pathways.

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