BACKGROUND Asiaticoside is a compound isolated from Herb Centella asiatica, which has been shown to promote osteogenic differentiation of human periodontal ligament (hPDL) cells. This study investigated the molecular mechanism underlying the asiaticoside-induced osteogenic differentiation of hPDL cells. METHODS hPDL cells were incubated with various concentrations of asiaticoside to test cell viability by MTT assay. The mRNA expression levels were analyzed by using quantitative real-time polymerase chain reaction (PCR). Osteogenic differentiation was determined by alkaline phosphatase activity assay and alizarin red staining. The subcellular localization of β-catenin was analyzed by both immunofluorescence and western blot. RESULTS The results showed that asiaticoside had no effect on the cell viability at any of the tested concentrations. Real-time PCR revealed that osterix (OSX) and dentin matrix protein1 (DMP1) mRNA were significantly enhanced by asiaticoside treatment. Alkaline phosphatase activity and in vitro mineralization were also significantly induced. Interestingly, asiaticoside dose-dependently increased WNT3A mRNA expression, but not WNT5A and WNT10B. The activation of Wnt signaling was shown to result in nuclear accumulation of β-catenin as evaluated by immunofluorescence staining and western blot analysis. Pre-treatment with recombinant human Dickkopf1 (rhDKK1) inhibited asiaticoside-induced β-catenin nuclear translocation and osteoblast marker gene expression. Moreover, rhDKK1 attenuated asiaticoside-induced DMP1 protein expression. CONCLUSION The data demonstrate that asiaticoside induces osteogenic differentiation of hPDL cells by activating the Wnt/β-catenin signaling pathway. The findings suggest that asiaticoside could be used as a novel therapeutic drug for periodontal tissue regeneration.

中文翻译：

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积雪草苷通过Wnt途径诱导人牙周膜细胞的成骨分化。

背景技术积雪草苷是从积雪草（Centella asiatica）中分离的化合物，已显示出它可促进人牙周膜（hPDL）细胞的成骨分化。这项研究调查了积雪草苷诱导的hPDL细胞成骨分化的分子机制。方法将hPDL细胞与不同浓度的积雪草苷一起孵育，通过MTT法检测细胞活力。使用定量实时聚合酶链反应（PCR）分析mRNA表达水平。通过碱性磷酸酶活性测定和茜素红染色确定成骨分化。通过免疫荧光和蛋白质印迹分析β-catenin的亚细胞定位。结果结果表明，积雪草苷在任何测试浓度下均不影响细胞活力。实时PCR显示，积雪草苷处理可显着增强osterix（OSX）和牙本质基质蛋白1（DMP1）mRNA。碱性磷酸酶活性和体外矿化也被明显诱导。有趣的是，积雪草苷剂量依赖性地增加了WNT3A mRNA的表达，但没有增加WNT5A和WNT10B的表达。如通过免疫荧光染色和蛋白质印迹分析所评估的，显示出Wnt信号传导的激活导致β-连环蛋白的核积累。重组人Dickkopf1（rhDKK1）的预处理抑制积雪草苷诱导的β-catenin核移位和成骨细胞标志物基因表达。此外，rhDKK1减弱积雪草苷诱导的DMP1蛋白表达。结论数据表明积雪草苷通过激活Wnt /β-catenin信号传导途径诱导hPDL细胞的成骨分化。这些发现表明积雪草苷可以用作牙周组织再生的新型治疗药物。