Abstract
BackgroundIdentifying bone anabolic agents is a superior strategy for the treatment of osteoporosis. Naturally, derived coumarin derivatives have shown osteoanabolic effect in vitro and in vivo. In this study, we investigated the effect of 5′-Hydroxy Auraptene (5′-HA), a coumarin derivative that newly isolated from Lotus lalambensis Schweinf on the differentiation of the mouse bone marrow-derived mesenchymal (skeletal) stem cells (mBMSCs) into osteoblast and adipocyte.MethodsThe effect of 5′-HA on mBMSCs cell proliferation and osteoblast differentiation was assessed by measuring cell viability, quantitative alkaline phosphatase (ALP) activity assay, Alizarin red staining for matrix mineralization and osteogenic gene array expression. Adipogenesis was measured by Oil Red O staining and quantitative real time PCR (qPCR) analysis of adipogenic markers. Regulation of BMPs signaling pathways by 5′-HA was measured by Western blot analysis and qPCR.Results5′-HA showed to stimulate the differentiation of mBMSCs into osteogenic cell lineage in a dose-dependent manner, without affecting their differentiation into adipocytic cell lineage. Treatment of mBMSCs with 5′-HA showed to promote significantly the BMP2-induced osteogenesis in mBMSCs via activating Smad1/5/8 phosphorylation and increasing Smad4 expression. Blocking of BMP signaling using BMPR1 selective inhibitor LDN-193189 significantly inhibited the stimulatory effect of 5′-HA on osteogenesis.ConclusionsOur data identified 5′-HA, as a novel coumarin derivative that function to stimulate the differentiation of mBMSCs into osteoblasts in BMP-signaling dependent mechanism.
Highlights
Osteoporosis is an endocrine-metabolic bone disease, characterized by reduced bone mass, strength, and microarchitecture, which increases the propensity of fragility fractures [1]
Cytotoxicity of 5′-Hydroxy Auraptene (5′-HA) on cultured primary Mouse bone marrow-derived mesenchymal stem cells (mBMSCs) as measured by MMT assay (a) and Cell Titer-Blue® cell viability assay (b) after 3 days in culture. c Effect of 5′-HA (1–50 μM) on cell proliferation of cultured mBMSCs as measured by counting the cell number at different concentrations of 5′-HA after 5 and 10 days. d Dose-dependent stimulatory effect of 5′-HA on osteoblast differentiation of mBMSCs as measured by quantification of alkaline phosphatase (ALP) activity after 7 days of induction and (e) quantitative Alizarin red staining for matrix mineralization after 12 days of induction
5′-HA exerts no effect on the differentiation of mBMSCs into adipocytes Since osteoblasts and adipocytes in bone marrow are derived from the same mBMSCs [6], we studied the effect of 5′-HA on the differentiation of mBMSCs into adipocytes. 5′-HA at different concentrations did not affect the adipogenesis of mBMSCs as revealed by quantification of Oil Red O staining for lipid accumulation (Fig. 3a)
Summary
The effect of 5′-HA on mBMSCs cell proliferation and osteoblast differentiation was assessed by measuring cell viability, quantitative alkaline phosphatase (ALP) activity assay, Alizarin red staining for matrix mineralization and osteogenic gene array expression. Adipogenesis was measured by Oil Red O staining and quantitative real time PCR (qPCR) analysis of adipogenic markers. Regulation of BMPs signaling pathways by 5′-HA was measured by Western blot analysis and qPCR. Results: 5′-HA showed to stimulate the differentiation of mBMSCs into osteogenic cell lineage in a dose-dependent manner, without affecting their differentiation into adipocytic cell lineage. Treatment of mBMSCs with 5′-HA showed to promote significantly the BMP2-induced osteogenesis in mBMSCs via activating Smad1/5/8 phosphorylation and increasing Smad expression. Blocking of BMP signaling using BMPR1 selective inhibitor LDN-193189 significantly inhibited the stimulatory effect of 5′-HA on osteogenesis
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