Purpose: Puerarin (C₂₁H₂₀O₁₀) is a phytoestrogen that possesses various pharmacological effect, and several researches have revealed the relationship between puerarin and bone metabolism. This study was aimed to evaluate the potential influence of puerarin on the proliferation and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) as well as on new bone formation following rapid maxillary expansion (RME) model in rats.
Methods: Rat BMSCs were adopted, and the cell proliferation was detected by cell-counting kit-8 (CCK-8) assay in vitro experiments. Alkaline phosphatase (ALP) activity and alizarin red staining were analyzed quantitatively to show extracellular matrix mineralization. The mRNA and protein expression levels were used to detect osteogenic differentiation of BMSCs. In vivo bone regeneration was analyzed in a rat RME model. Eighteen 6-week-old male Wistar rats were divided into 3 groups: group 1 without any treatment, group 2 received RME and saline solution (15mg/kg), group 3 received RME and puerarin solution (15mg/kg). After 2 weeks, micro-computed tomography (Micro-CT), hematoxylin and eosin (HE) staining, and Masson staining were used to detect the new bone formation and morphological changes. Besides, ALP and bone morphogenetic protein 2 (BMP2) expression levels in mid-palatal suture were evaluated by immunohistochemical staining.
Results: The results showed that puerarin upregulates cell proliferation dose-dependently. ALP activity and mineralized matrix generation were clearly enhanced at certain specific concentrations (10^{− 5} and 10^{− 6} mol/L); the expression levels of the osteoblast-related genes and proteins were increased. The measurement of micro-CT imaging revealed that puerarin significantly promoted new bone formation. Concomitantly, the histological examinations showed that puerarin solution enhanced osteogenesis in mid-palatal suture.
Conclusion: Those works indicated that puerarin regulates osteogenesis in vitro and exerts a beneficial impact on bone regeneration in vivo, revealing that puerarin treatment may become one of the potential keys for improving the stability and preventing relapse of RME.

Keywords: bone marrow-derived mesenchymal stem cells, bone regeneration, micro-CT, rapid maxillary expansion


中文翻译：

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葛根素对体外成骨分化和体内新骨形成的影响

目的：葛根素（C ₂₁ H ₂₀ O ₁₀）是一种具有多种药理作用的植物雌激素，多项研究揭示了葛根素与骨代谢的关系。本研究旨在评估葛根素对大鼠骨髓间充质干细胞 (BMSCs) 增殖和成骨分化以及大鼠快速上颌扩张 (RME) 模型后新骨形成的潜在影响。
方法：采用大鼠骨髓间充质干细胞，体外实验采用细胞计数试剂盒8（CCK-8）法检测细胞增殖情况。定量分析碱性磷酸酶 (ALP) 活性和茜素红染色以显示细胞外基质矿化。mRNA和蛋白表达水平用于检测骨髓间充质干细胞的成骨分化。在大鼠 RME 模型中分析体内骨再生。18只6周龄雄性Wistar大鼠分为3组：第1组不做任何处理，第2组接受RME和生理盐水（15mg/kg），第3组接受RME和葛根素溶液（15mg/kg）。2周后采用微计算机断层扫描（Micro-CT）、苏木精和伊红（HE）染色、Masson染色检测新骨形成及形态变化。除了，
结果：结果显示葛根素上调细胞增殖呈剂量依赖性。^{在某些特定浓度（10 - 5}和 10 ^{- 6} mol/L）下， ALP 活性和矿化基质生成明显增强；成骨细胞相关基因和蛋白质的表达水平增加。显微CT成像测量显示葛根素显着促进新骨形成。同时，组织学检查显示葛根素溶液增强了腭中缝的成骨。
结论：这些工作表明葛根素在体外调节成骨，对体内骨再生产生有益的影响，表明葛根素治疗可能成为提高RME稳定性和预防复发的潜在关键之一。

关键词：骨髓间充质干细胞，骨再生，显微CT，快速上颌扩张