Stimulatory effect of puerarin on bone formation through co-activation of nitric oxide and bone morphogenetic protein-2/mitogen-activated protein kinases pathways in mice.
BACKGROUND
Estrogen deficiency results in loss of bone mass. Phytoestrogens are plant-derived non-steroidal compounds with estrogen-like activity that bind to estrogen receptors. The main aim of this study was to investigate the effect of the phytoestrogen puerarin on adult mouse osteoblasts.


METHODS
Osteoblast cells were harvested from 8-month old female imprinting control region (ICR) mice. The effects of puerarin stimulation on the proliferation, differentiation and maturation of osteoblasts were examined. The production of nitric oxide (NO) and the expression of bone morphogenetic protein-2 (BMP-2), SMAD4, mitogen-activated protein kinases (MAPK), core binding factor α1/ runt-related transcription factor 2 (Cbfa1/Runx2), osteoprotegerin (OPG), and receptor activator of NF-κB ligand (RANKL) genes were analyzed. The activation of signal pathways was further confirmed by specific pathway inhibitors.


RESULTS
The osteoblast viability reached its maximum at 10(-8) mol/L puerarin. At this concentration, puerarin increases the proliferation and matrix mineralization of osteoblasts and promotes NO synthesis. With 10(-8) mol/L puerarin treatment, BMP-2, SMAD4, Cbfa1/Runx2, and OPG gene expression were up-regulated, while the RANKL gene expression is down-regulated. Concurrent treatment involving the (bone morphogenetic protein) BMP antagonist Noggin or the NOS inhibitor L-NAME diminishes puerarin induced cell proliferation, Alkaline phosphatase (ALP) activity, NO production, as well as the BMP-2, SMAD4, Cbfa1/Runx2, OPG, and RANKL gene expression.


CONCLUSIONS
In this in vitro study, we demonstrate that puerarin is a bone anabolic agent that exerts its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating Cbfa1/Runx2, OPG, and RANKL gene expression. This effect may contribute to its induction of osteoblast proliferation and differentiation, resulting in bone formation.
School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan, China. amel@tmu.edu.tw
Shiow-Yunn
SY
Sheu
Chia-Chung
CC
Tsai
Jui-Sheng
JS
Sun
Ming-Hong
MH
Chen
Man-Hai
MH
Liu
Man-Ger
MG
Sun
D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005260:Female; D007529:Isoflavones / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D008813:Mice, Inbred ICR; D009569:Nitric Oxide / Q000502:physiology*; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects*; D048789:Phytoestrogens / Q000494:pharmacology*; D053245:RANK Ligand / Q000235:genetics
D055396:Bone Morphogenetic Protein 2; D007529:Isoflavones; D048789:Phytoestrogens; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse; D009569:Nitric Oxide; C033607:puerarin