Composition of BSHXF.

BackgroundThe BushenHuoxue formula (BSHX) has been previously demonstrated to ameliorate osteoporosis, but the mechanisms underlying this phenomenon are currently unclear. The present study aims at investigating the mechanisms that BSHX induces osteogenesis.MethodsWe established an osteoporosis model in rats by bilateral ovariectomy and then treated the rats with an osteogenic inducer (dexamethasone, β-sodium glycerophosphate and Vitamin C) and BSHX. After that, bone marrow density and histopathological bone examination were evaluated by using HE staining and immunohistochemistry, respectively. We also assessed the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts by using immunofluorescence staining. ALP, BMP, and COL1A1 levels were determined by ELISA. We identified genes involved in pathogenesis of osteoporosis through Gene Expression Omnibus (GEO) database and subsequently selected Hedgehog signaling-related genes Shh, Ihh, Gli2, and Runx2 for assessment via qRT-PCR and ELISA, Western blotting. Network pharmacology analysis was performed to identify bioactive metabolites of BSHX.ResultsBSHX treatment in osteoporosis model rats promoted tightening of the morphological structure of the trabecular bone and increased the bone mineral density (BMD). BSHX also increased levels of osteoblast makers ALP, BMP, and COL1A1. Additionally, bioinformatics analysis of the GEO dataset showed that Hedgehog signaling pathway was involved in pathogenesis of osteoporosis, especially related genes Shh, Ihh, Gli2, and Runx2. Remarkably, BHSX upregulated these genes indispensably involved in the osteogenesis-related Hedgehog signaling pathway in both bone tissue and BMSCs. Importantly, we identified that quercetin was the active compounds that involved in the mechanism of BSHX-improved OP via affecting Hedgehog-related genes.ConclusionOur results indicate that BSHX promotes osteogenesis by improving BMSC differentiation into osteoblasts via increased expression of Hedgehog signaling-related genes Shh, Ihh, Gli2, and Runx2, and quercetin was the bioactive compound of BSHX.背景 补肾活血方（BushenHuoxue formula, BSHX）既往已被证实可改善骨质疏松症，但该作用的具体分子机制目前尚不明确。本研究旨在探讨补肾活血方诱导成骨的潜在作用机制。 方法 本研究通过双侧卵巢切除术构建大鼠骨质疏松模型，随后分别使用成骨诱导剂（地塞米松、β-甘油磷酸钠与维生素C）及补肾活血方对大鼠进行干预。随后分别采用HE染色与免疫组化法检测大鼠骨密度（bone mineral density, BMD）与骨组织病理形态；通过免疫荧光染色法评估骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）向成骨细胞的分化情况。采用ELISA法检测碱性磷酸酶（alkaline phosphatase, ALP）、骨形态发生蛋白（bone morphogenetic protein, BMP）及Ⅰ型胶原α1链（collagen type I alpha 1 chain, COL1A1）的表达水平。通过基因表达综合数据库（Gene Expression Omnibus, GEO）筛选与骨质疏松发病机制相关的基因，随后选取刺猬信号通路（Hedgehog signaling pathway）相关基因Shh、Ihh、Gli2及Runx2，采用实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）、ELISA与蛋白质印迹法（Western blotting）进行检测。采用网络药理学分析方法筛选补肾活血方的活性代谢产物。 结果 骨质疏松模型大鼠经补肾活血方干预后，骨小梁形态结构更加紧密，骨密度显著升高。补肾活血方还可上调成骨细胞标志物ALP、BMP及COL1A1的表达水平。此外，对GEO数据集的生物信息学分析显示，刺猬信号通路参与了骨质疏松的发病过程，尤其与Shh、Ihh、Gli2及Runx2等相关基因密切相关。值得注意的是，补肾活血方能显著上调骨组织与BMSCs中参与成骨相关刺猬信号通路的上述关键基因的表达。更为重要的是，本研究证实槲皮素是补肾活血方通过调控刺猬信号通路相关基因改善骨质疏松的核心活性成分。 结论 本研究结果表明，补肾活血方通过上调刺猬信号通路相关基因Shh、Ihh、Gli2及Runx2的表达，促进骨髓间充质干细胞向成骨细胞分化，从而发挥成骨作用；且槲皮素为补肾活血方发挥药效的核心活性成分。