Department of Oral and Maxillofacial Pathology, School of Dentistry, Kyung Hee University, Seoul 02447, Korea.
National Institute for Korean Medicine Development, Gyeongsan 38540, Korea.
Int J Mol Sci. 2020 Jul 27;21(15):5332. doi: 10.3390/ijms21155332.
Plant extracts have long been regarded as useful medicines in the treatment of human diseases. Nakai has been used as a traditional medicine, as it has pharmacological properties such as antioxidant and anti-inflammatory activity. However, the biological functions of TMARg, isolated from the roots of in osteoblast differentiation remain unknown. This study was performed to investigate the pharmacological effects and intracellular signaling of TMARg in the osteoblast differentiation of pre-osteoblast MC3T3-E1 cells and mesenchymal precursor C2C12 cells.
Cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP), and by staining it with Alizarin red S (ARS). Cell migration was determined by using migration assays. Western blot analysis and immunocytochemical analysis were used to examine the intracellular signaling pathways and differentiation proteins.
In the present study, TMARg showed no cytotoxicity and increased the osteoblast differentiation in pre-osteoblasts, as assessed from the alkaline phosphate (ALP) staining and activity and ARS staining. TMARg also induced BMP2 expression and increased the p-smad1/5/8-RUNX2 and β-catenin pathways in both MC3T3-E1 and C2C12 cells. Furthermore, TMARg activated mitogen-activated protein kinases (MAPKs) and increased the cell migration rate. In addition, the TMARg-mediated osteoblast differentiation was suppressed by BMP and Wnt inhibitors with the downregulation of BMP2 expression.
These findings demonstrate that TMARg exerts pharmacological and biological effects on osteoblast differentiation through the activation of BMP2 and β-catenin signaling pathways, and suggest that TMARg might be a potential phytomedicine for the treatment of bone diseases.
植物提取物长期以来一直被认为是治疗人类疾病的有用药物。Nakai 作为一种传统药物，具有抗氧化和抗炎活性等药理学特性。然而，从 根部分离的 TMARg 在成骨细胞分化中的生物学功能尚不清楚。本研究旨在探讨 TMARg 在成骨前体细胞 MC3T3-E1 细胞和间充质前体细胞 C2C12 细胞中的成骨细胞分化中的药理作用和细胞内信号转导。
通过 MTT 测定评估细胞活力。通过分析碱性磷酸酶（ALP）的活性并用茜素红 S（ARS）染色来检测早期和晚期成骨细胞分化。通过迁移测定确定细胞迁移。使用 Western blot 分析和免疫细胞化学分析来检查细胞内信号通路和分化蛋白。
在本研究中，TMARg 没有显示出细胞毒性，并通过碱性磷酸酶（ALP）染色和活性以及 ARS 染色增加了成骨前体细胞的成骨细胞分化。TMARg 还诱导了 BMP2 的表达，并增加了 MC3T3-E1 和 C2C12 细胞中的 p-smad1/5/8-RUNX2 和 β-catenin 途径。此外，TMARg 激活丝裂原活化蛋白激酶（MAPKs）并增加细胞迁移率。此外，BMP 和 Wnt 抑制剂下调 BMP2 表达，抑制了 TMARg 介导的成骨细胞分化。
这些发现表明，TMARg 通过激活 BMP2 和 β-catenin 信号通路对成骨细胞分化发挥药理和生物学作用，并表明 TMARg 可能是治疗骨疾病的潜在植物药。
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