Abstract
Dalbergia odorifera has been traditionally used as a medicine to treat many diseases. However, the role of 2,4,5-trimethoxyldalbergiquinol (TMDQ) isolated and extracted from D. odorifera in osteoblast function and the underlying molecular mechanisms remain poorly understood. The aim of this study was to investigate the effects and possible underlying mechanisms of TMDQ on osteoblastic differentiation of primary cultures of mouse osteoblasts as an in vitro assay system. TMDQ stimulated osteoblastic differentiation, as assessed by the alkaline phosphatase (ALP) activity, ALP staining, mineralized nodule formation, and the levels of mRNAs encoding the bone differentiation markers, including ALP, bone sialoprotein (BSP), osteopontin, and osteocalcin. TMDQ upregulated the expression of Bmp2 and Bmp4 genes, and increased the protein level of phospho-Smad1/5/8. Furthermore, TMDQ treatment showed the increased mRNA expression of Wnt ligands, phosphorylation of GSK3, and the expression of β-catenin protein. The TMDQ-induced osteogenic effects were abolished by Wnt inhibitor, Dickkopf-1 (DKK1), and bone morphogenetic protein (BMP) antagonist, noggin. TMDQ-induced runt-related transcription factor 2 (Runx2) expression was attenuatted by noggin and DKK1. These data suggest that TMDQ acts through the activation of BMP, Wnt/β-catenin, and Runx2 signaling to promote osteoblast differentiation, and we demonstrate that TMDQ could be a potential agent for the treatment of bone loss-associated diseases such as osteoporosis.
Highlights
Potent anticatabolic drugs that include estrogen, bisphosphonates, and RANKL-inhibitor have been the main therapies for osteoporosis.[2]
To understand the signal pathways involved in the regulation of cell differentiation by TMDQ, mitogen-activated protein kinase (MAPK), bone morphogenetic protein (BMP), and Wnt/β-catenin pathways were analyzed via reverse transcriptase-PCR (RT-PCR), real-time PCR, western blot analysis, and immunofluorescence
To examine the link between the activation of the BMP and Wnt/β-catenin pathways, we investigated the effects of TMDQ on β-catenin downstream target gene, runt-related transcription factor 2 (Runx2), which is a key transcription factor that has an essential role in osteoblastic differentiation.[14]
Summary
Potent anticatabolic drugs that include estrogen, bisphosphonates, and RANKL-inhibitor have been the main therapies for osteoporosis.[2]. Previous phytochemical studies reported that the isolated phenolic compounds of D. odorifera such as isoflavanones, isoflavans, neoflavonoids, and chalcones exert various beneficial properties, such as anti-oxidant, anti-microbial, and anti-inflammatory effects.[5,6,7,8,9,10] We previously reported that 4,2′,5′-trihydroxy-4′-methoxychalcone, a biologically active chalcone isolated from the heartwood of D. odorifera, exhibited protective effects against glutamate-induced oxidative injury in HT22 cells.[10] In addition, we demonstrated that 4,2′,5′-trihydroxy-4′-methoxychalcone exhibits antiinflammatory properties by inducing heme oxygenase-1 in murine macrophages.[11]. We isolated and identified novel compound 2,4,5-trimethoxyldalbergiquinol (TMDQ) compound from the heartwood of D. odorifera, its pharmacological effects in bone-forming cells have not been defined yet. The aim of this work was to investigate the effects and its underlying signal mechanism of TMDQ on osteoblastic differentiation in primary culture of mouse calvarial osteoblasts as an in vitro assay system
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