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Flavonoid compound icariin enhances BMP-2 induced differentiation and signalling by targeting to connective tissue growth factor (CTGF) in SAMP6 osteoblasts

BACKGROUND: Icariin, a major active flavonoid glucoside, is widely used for the treatment of bone injury and rebuilding in the clinic because of its roles in suppressing osteoblastogenesis and promoting osteogenesis. The senescence-accelerated mouse SAMP6 was accepted as a useful murine model to rev...

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Autores principales: Xu, Bing, Wang, Xueqiang, Wu, Chengliang, Zhu, Lihe, Chen, Ou, Wang, Xiaofeng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039035/
https://www.ncbi.nlm.nih.gov/pubmed/29990327
http://dx.doi.org/10.1371/journal.pone.0200367
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author Xu, Bing
Wang, Xueqiang
Wu, Chengliang
Zhu, Lihe
Chen, Ou
Wang, Xiaofeng
author_facet Xu, Bing
Wang, Xueqiang
Wu, Chengliang
Zhu, Lihe
Chen, Ou
Wang, Xiaofeng
author_sort Xu, Bing
collection PubMed
description BACKGROUND: Icariin, a major active flavonoid glucoside, is widely used for the treatment of bone injury and rebuilding in the clinic because of its roles in suppressing osteoblastogenesis and promoting osteogenesis. The senescence-accelerated mouse SAMP6 was accepted as a useful murine model to reveal the mechanism of senile osteoporosis and the therapeutic mechanism of drug activity. However, little is known about the characteristics of SAMP6 osteoblasts and the associated regulatory roles of icariin. METHODS: We isolated and cultured osteoblasts from SAMP6 or SAMR1 mice and compared their proliferation, migration, and differentiation by performing the CCK-8 assay, cell counting assay, EdU staining, cell cycle analysis, ALP staining and activity measurement, Alizarin red staining, and RT-qPCR analysis to measure the levels of osteoblast markers, including RUNX2, Colla1 and Oc. To assess the effects of icariin on BMP-2-induced osteoblast differentiation, after BMP-2 treatment, osteoblast markers were analyzed by RT-qPCR and semi-quantitative Western blotting. The effects of icariin on connective tissue growth factor (CTGF) were measured by RT-qPCR. shRNA targeting CTGF mRNA was employed to knockdown its expression level in osteoblasts. RESULTS: The SAMP6 osteoblasts presented decreased the development and differentiation activity compared with SAMR1 osteoblasts, indicating that they are the potential mechanisms underlying age-associated disease. Moreover, SAMP6 osteoblasts presented upregulated CTGF compared with SAMR1 osteoblasts. Icariin enhanced BMP-2-induced osteoblast differentiation by downregulating CTGF expression, which tightly regulates osteoblast differentiation. By downregulating CTGF, icariin treatment upregulated phosphate-Smad1/5/8, indicating its activating effects on the BMP signaling pathway. CONCLUSION: These results suggest that decreased osteoblast development and function potentially contributes to age-associated disease. Icariin exerts enhancing effects on BMP-2-mediated osteoblast development via downregulating CTGF.
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spelling pubmed-60390352018-07-19 Flavonoid compound icariin enhances BMP-2 induced differentiation and signalling by targeting to connective tissue growth factor (CTGF) in SAMP6 osteoblasts Xu, Bing Wang, Xueqiang Wu, Chengliang Zhu, Lihe Chen, Ou Wang, Xiaofeng PLoS One Research Article BACKGROUND: Icariin, a major active flavonoid glucoside, is widely used for the treatment of bone injury and rebuilding in the clinic because of its roles in suppressing osteoblastogenesis and promoting osteogenesis. The senescence-accelerated mouse SAMP6 was accepted as a useful murine model to reveal the mechanism of senile osteoporosis and the therapeutic mechanism of drug activity. However, little is known about the characteristics of SAMP6 osteoblasts and the associated regulatory roles of icariin. METHODS: We isolated and cultured osteoblasts from SAMP6 or SAMR1 mice and compared their proliferation, migration, and differentiation by performing the CCK-8 assay, cell counting assay, EdU staining, cell cycle analysis, ALP staining and activity measurement, Alizarin red staining, and RT-qPCR analysis to measure the levels of osteoblast markers, including RUNX2, Colla1 and Oc. To assess the effects of icariin on BMP-2-induced osteoblast differentiation, after BMP-2 treatment, osteoblast markers were analyzed by RT-qPCR and semi-quantitative Western blotting. The effects of icariin on connective tissue growth factor (CTGF) were measured by RT-qPCR. shRNA targeting CTGF mRNA was employed to knockdown its expression level in osteoblasts. RESULTS: The SAMP6 osteoblasts presented decreased the development and differentiation activity compared with SAMR1 osteoblasts, indicating that they are the potential mechanisms underlying age-associated disease. Moreover, SAMP6 osteoblasts presented upregulated CTGF compared with SAMR1 osteoblasts. Icariin enhanced BMP-2-induced osteoblast differentiation by downregulating CTGF expression, which tightly regulates osteoblast differentiation. By downregulating CTGF, icariin treatment upregulated phosphate-Smad1/5/8, indicating its activating effects on the BMP signaling pathway. CONCLUSION: These results suggest that decreased osteoblast development and function potentially contributes to age-associated disease. Icariin exerts enhancing effects on BMP-2-mediated osteoblast development via downregulating CTGF. Public Library of Science 2018-07-10 /pmc/articles/PMC6039035/ /pubmed/29990327 http://dx.doi.org/10.1371/journal.pone.0200367 Text en © 2018 Xu et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Xu, Bing
Wang, Xueqiang
Wu, Chengliang
Zhu, Lihe
Chen, Ou
Wang, Xiaofeng
Flavonoid compound icariin enhances BMP-2 induced differentiation and signalling by targeting to connective tissue growth factor (CTGF) in SAMP6 osteoblasts
title Flavonoid compound icariin enhances BMP-2 induced differentiation and signalling by targeting to connective tissue growth factor (CTGF) in SAMP6 osteoblasts
title_full Flavonoid compound icariin enhances BMP-2 induced differentiation and signalling by targeting to connective tissue growth factor (CTGF) in SAMP6 osteoblasts
title_fullStr Flavonoid compound icariin enhances BMP-2 induced differentiation and signalling by targeting to connective tissue growth factor (CTGF) in SAMP6 osteoblasts
title_full_unstemmed Flavonoid compound icariin enhances BMP-2 induced differentiation and signalling by targeting to connective tissue growth factor (CTGF) in SAMP6 osteoblasts
title_short Flavonoid compound icariin enhances BMP-2 induced differentiation and signalling by targeting to connective tissue growth factor (CTGF) in SAMP6 osteoblasts
title_sort flavonoid compound icariin enhances bmp-2 induced differentiation and signalling by targeting to connective tissue growth factor (ctgf) in samp6 osteoblasts
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039035/
https://www.ncbi.nlm.nih.gov/pubmed/29990327
http://dx.doi.org/10.1371/journal.pone.0200367
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