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A novel RANKL‐targeted flavonoid glycoside prevents osteoporosis through inhibiting NFATc1 and reactive oxygen species

BACKGROUND AND PURPOSE: Osteoporosis is characterized by excessive bone resorption due to enhanced osteoclast activation. Stimulation of nuclear factor of activated T cells 1 (NFATc1) and accumulation of reactive oxygen species (ROS) are important mechanisms underlying osteoclastogenesis. Robinin (R...

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Autores principales: Hong, Guoju, Chen, Zhenqiu, Han, Xiaorui, Zhou, Lin, Pang, Fengxiang, Wu, Rishana, Shen, Yingshan, He, Xiaoming, Hong, Zhinan, Li, Ziqi, He, Wei, Wei, Qiushi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140192/
https://www.ncbi.nlm.nih.gov/pubmed/34047464
http://dx.doi.org/10.1002/ctm2.392
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author Hong, Guoju
Chen, Zhenqiu
Han, Xiaorui
Zhou, Lin
Pang, Fengxiang
Wu, Rishana
Shen, Yingshan
He, Xiaoming
Hong, Zhinan
Li, Ziqi
He, Wei
Wei, Qiushi
author_facet Hong, Guoju
Chen, Zhenqiu
Han, Xiaorui
Zhou, Lin
Pang, Fengxiang
Wu, Rishana
Shen, Yingshan
He, Xiaoming
Hong, Zhinan
Li, Ziqi
He, Wei
Wei, Qiushi
author_sort Hong, Guoju
collection PubMed
description BACKGROUND AND PURPOSE: Osteoporosis is characterized by excessive bone resorption due to enhanced osteoclast activation. Stimulation of nuclear factor of activated T cells 1 (NFATc1) and accumulation of reactive oxygen species (ROS) are important mechanisms underlying osteoclastogenesis. Robinin (Rob) is a flavonoid glycoside that has shown anti‐inflammatory and antioxidative effects in previous studies, but little is known about its effects on bone homeostasis. The purpose of our research was to investigate whether Rob could prevent bone resorption in ovariectomized (OVX) mice by suppressing osteoclast production through its underlying mechanisms. METHODS: The docking pose of Rob and RANKL was identified by protein‐ligand molecular docking. Rob was added to bone marrow macrophages (BMMs) stimulated by nuclear factor‐κB (NF‐κB) ligand (RANKL). The effects of Rob on osteoclastic activity were evaluated by positive tartrate resistant acid phosphatase (TRAcP) staining kit and hydroxyapatite resorption assay. RANKL‐induced ROS generation in osteoclasts was detected by H(2)DCFDA and MitoSox Red staining. The classic molecular cascades triggered by RANKL, such as NF‐κB, ROS, calcium oscillations, and NFATc1‐mediated signaling pathways, were investigated using Fluo4 staining, western blot, and quantitative real‐time polymerase chain reaction. In addition, an OVX mouse model mimicking estrogen‐deficient osteoporosis was created to evaluate the therapeutic effects of Rob in vivo. RESULTS: Computational docking results showed that Rob could bind specifically to RANKL's predicted binding sites. In vitro, Rob inhibited RANKL‐mediated osteoclastogenesis dose‐dependently without obvious cytotoxicity at low concentrations. We also found that Rob attenuated RANKL‐induced mitochondrial ROS production or enhanced activities of ROS‐scavenging enzymes, and ultimately reduced intracellular ROS levels. Rob abrogated the RANKL‐induced mitogen‐activated protein kinase (MAPK) and NF‐κB signaling pathways, and subsequently blocked NFATc1 signaling and TRAcP expression. In addition, Rob inhibited osteoclast proliferation by downregulating the expression of osteoclast target genes (Acp5, Cathepsin K, Atp6v0d2, Nfact1, c‐Fos, and Mmp9) and reducing Ca(2+) oscillations. Our in vivo results showed that Rob reduced bone resorption in OVX animal model by repressing osteoclast activity and function. CONCLUSIONS: Rob inhibits the activation of osteoclasts by targeting RANKL and is therefore a potential osteoporosis drug.
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spelling pubmed-81401922021-05-26 A novel RANKL‐targeted flavonoid glycoside prevents osteoporosis through inhibiting NFATc1 and reactive oxygen species Hong, Guoju Chen, Zhenqiu Han, Xiaorui Zhou, Lin Pang, Fengxiang Wu, Rishana Shen, Yingshan He, Xiaoming Hong, Zhinan Li, Ziqi He, Wei Wei, Qiushi Clin Transl Med Research Articles BACKGROUND AND PURPOSE: Osteoporosis is characterized by excessive bone resorption due to enhanced osteoclast activation. Stimulation of nuclear factor of activated T cells 1 (NFATc1) and accumulation of reactive oxygen species (ROS) are important mechanisms underlying osteoclastogenesis. Robinin (Rob) is a flavonoid glycoside that has shown anti‐inflammatory and antioxidative effects in previous studies, but little is known about its effects on bone homeostasis. The purpose of our research was to investigate whether Rob could prevent bone resorption in ovariectomized (OVX) mice by suppressing osteoclast production through its underlying mechanisms. METHODS: The docking pose of Rob and RANKL was identified by protein‐ligand molecular docking. Rob was added to bone marrow macrophages (BMMs) stimulated by nuclear factor‐κB (NF‐κB) ligand (RANKL). The effects of Rob on osteoclastic activity were evaluated by positive tartrate resistant acid phosphatase (TRAcP) staining kit and hydroxyapatite resorption assay. RANKL‐induced ROS generation in osteoclasts was detected by H(2)DCFDA and MitoSox Red staining. The classic molecular cascades triggered by RANKL, such as NF‐κB, ROS, calcium oscillations, and NFATc1‐mediated signaling pathways, were investigated using Fluo4 staining, western blot, and quantitative real‐time polymerase chain reaction. In addition, an OVX mouse model mimicking estrogen‐deficient osteoporosis was created to evaluate the therapeutic effects of Rob in vivo. RESULTS: Computational docking results showed that Rob could bind specifically to RANKL's predicted binding sites. In vitro, Rob inhibited RANKL‐mediated osteoclastogenesis dose‐dependently without obvious cytotoxicity at low concentrations. We also found that Rob attenuated RANKL‐induced mitochondrial ROS production or enhanced activities of ROS‐scavenging enzymes, and ultimately reduced intracellular ROS levels. Rob abrogated the RANKL‐induced mitogen‐activated protein kinase (MAPK) and NF‐κB signaling pathways, and subsequently blocked NFATc1 signaling and TRAcP expression. In addition, Rob inhibited osteoclast proliferation by downregulating the expression of osteoclast target genes (Acp5, Cathepsin K, Atp6v0d2, Nfact1, c‐Fos, and Mmp9) and reducing Ca(2+) oscillations. Our in vivo results showed that Rob reduced bone resorption in OVX animal model by repressing osteoclast activity and function. CONCLUSIONS: Rob inhibits the activation of osteoclasts by targeting RANKL and is therefore a potential osteoporosis drug. John Wiley and Sons Inc. 2021-05-21 /pmc/articles/PMC8140192/ /pubmed/34047464 http://dx.doi.org/10.1002/ctm2.392 Text en © 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Articles
Hong, Guoju
Chen, Zhenqiu
Han, Xiaorui
Zhou, Lin
Pang, Fengxiang
Wu, Rishana
Shen, Yingshan
He, Xiaoming
Hong, Zhinan
Li, Ziqi
He, Wei
Wei, Qiushi
A novel RANKL‐targeted flavonoid glycoside prevents osteoporosis through inhibiting NFATc1 and reactive oxygen species
title A novel RANKL‐targeted flavonoid glycoside prevents osteoporosis through inhibiting NFATc1 and reactive oxygen species
title_full A novel RANKL‐targeted flavonoid glycoside prevents osteoporosis through inhibiting NFATc1 and reactive oxygen species
title_fullStr A novel RANKL‐targeted flavonoid glycoside prevents osteoporosis through inhibiting NFATc1 and reactive oxygen species
title_full_unstemmed A novel RANKL‐targeted flavonoid glycoside prevents osteoporosis through inhibiting NFATc1 and reactive oxygen species
title_short A novel RANKL‐targeted flavonoid glycoside prevents osteoporosis through inhibiting NFATc1 and reactive oxygen species
title_sort novel rankl‐targeted flavonoid glycoside prevents osteoporosis through inhibiting nfatc1 and reactive oxygen species
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140192/
https://www.ncbi.nlm.nih.gov/pubmed/34047464
http://dx.doi.org/10.1002/ctm2.392
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