Abstract：
BACKGROUND: Postmenopausal osteoporosis (PMOP) has been considered as a major causative factor for bone-joint pain and inducing pathologic fractures. Bu-Sui-Dan (BSD), a classic ancient herbal formula, has been shown to exhibit osteoprotective effects by promoting bone marrow development and bone growth. However, the exact mechanism of BSD are still unexplored.
OBJECTIVE: The study aimed to investigate the protective effect of BSD against osteoporotic injury, and to explore whether BSD regulated BMSCs\' osteogenic differentiation by targeting VGLL4, which in turn improved PMOP.
METHODS: The anti-osteoporotic effect of BSD was studied in ovariectomized (OVX) rats and bone marrow mesenchymal stem cells (BMSCs). Micro-CT imaging and HE staining were performed, and the levels of osteogenic protein RUNX2 and osteogenesis-related factor VGLL4 were determined. Co-immunoprecipitation (Co-IP) was further employed to delve into the effects of BSD on the interactions between TEAD4 and RUNX2. The key osteogenic factors 1ALP, COLl1A1, and Osterix expression were detected by RT-qPCR. Co-IP and proximity ligation assay (PLA) were employed to scrutinize the influence of BSD on TEAD4 and RUNX2 inter-binding. Moreover, VGLL4 knockdown in BMSCs was conducted to confirm the role of VGLL4 in the therapeutic mechanism of BSD.
RESULTS: BSD showed a dose-dependent protective effect against osteoporotic injury, as evidenced by improvement in bone volume, bone microarchitecture, and histomorphometry. Additionally, BSD treatment increased the levels of RUNX2 and its downstream target genes including ALP, COL1A1, and Osterix. Moreover, BSD upregulated VGLL4 expression and lessened TEAD4-RUNX2 interactions. In BMSCs experiment, BSD-containing serum could promote osteogenic differentiation of BMSCs, boosted the expression of osteogenesis-related factors and VGLL4 level. The knockdown of VGLL4 in BMSCs diminished the promotion effect of BSD in osteoblast differentiation, suggesting that VGLL4 play a vital role in the therapeutic effects exerted by BSD.
CONCLUSIONS: BSD ameliorated osteoporosis injury and promoted osteoblast differentiation through upregulation of VGLL4 levels, which in turn antagonized TEAD4-mediated RUNX2 transcriptional repression. Our study implied that BSD may be an osteoporosis therapeutic agent.
OBJECTIVE: The study aimed to investigate the protective effect of BSD against osteoporotic injury, and to explore whether BSD regulated BMSCs\' osteogenic differentiation by targeting VGLL4, which in turn improved PMOP.
METHODS: The anti-osteoporotic effect of BSD was studied in ovariectomized (OVX) rats and bone marrow mesenchymal stem cells (BMSCs). Micro-CT imaging and HE staining were performed, and the levels of osteogenic protein RUNX2 and osteogenesis-related factor VGLL4 were determined. Co-immunoprecipitation (Co-IP) was further employed to delve into the effects of BSD on the interactions between TEAD4 and RUNX2. The key osteogenic factors 1ALP, COLl1A1, and Osterix expression were detected by RT-qPCR. Co-IP and proximity ligation assay (PLA) were employed to scrutinize the influence of BSD on TEAD4 and RUNX2 inter-binding. Moreover, VGLL4 knockdown in BMSCs was conducted to confirm the role of VGLL4 in the therapeutic mechanism of BSD.
RESULTS: BSD showed a dose-dependent protective effect against osteoporotic injury, as evidenced by improvement in bone volume, bone microarchitecture, and histomorphometry. Additionally, BSD treatment increased the levels of RUNX2 and its downstream target genes including ALP, COL1A1, and Osterix. Moreover, BSD upregulated VGLL4 expression and lessened TEAD4-RUNX2 interactions. In BMSCs experiment, BSD-containing serum could promote osteogenic differentiation of BMSCs, boosted the expression of osteogenesis-related factors and VGLL4 level. The knockdown of VGLL4 in BMSCs diminished the promotion effect of BSD in osteoblast differentiation, suggesting that VGLL4 play a vital role in the therapeutic effects exerted by BSD.
CONCLUSIONS: BSD ameliorated osteoporosis injury and promoted osteoblast differentiation through upregulation of VGLL4 levels, which in turn antagonized TEAD4-mediated RUNX2 transcriptional repression. Our study implied that BSD may be an osteoporosis therapeutic agent.
摘要：
背景：绝经后骨质疏松症（PMOP）被认为是骨-关节痛和引起病理性骨折的主要原因。Bu-Sui-Dan(BSD),经典的古代草药配方，已显示通过促进骨髓发育和骨骼生长而表现出骨保护作用。然而,BSD的确切机制仍未被探索。
目的：本研究旨在探讨BSD对骨质疏松损伤的保护作用。并探讨BSD是否通过靶向VGLL4调节BMSCs的成骨分化，进而改善PMOP。
方法：在去卵巢（OVX）大鼠和骨髓间充质干细胞（BMSCs）中研究BSD的抗骨质疏松作用。进行Micro-CT成像和HE染色，并测定成骨蛋白RUNX2和成骨相关因子VGLL4的水平。进一步采用免疫共沉淀(Co-IP)来研究BSD对TEAD4和RUNX2之间的相互作用的影响。关键成骨因子1ALP,通过RT-qPCR检测COLl1A1和Osterix的表达。采用Co-IP和邻近连接测定(PLA)来仔细检查BSD对TEAD4和RUNX2相互结合的影响。此外，在BMSCs中进行VGLL4敲低以证实VGLL4在BSD的治疗机制中的作用。
结果：BSD对骨质疏松损伤具有剂量依赖性保护作用，骨体积的改善证明了这一点，骨微结构，和组织形态计量学。此外,BSD处理增加了RUNX2及其下游靶基因包括ALP的水平,COL1A1和Osterix.此外，BSD上调VGLL4表达并减少TEAD4-RUNX2相互作用。在BMSCs实验中,含BSD的血清能促进BMSCs成骨分化,增强成骨相关因子的表达和VGLL4水平。BMSCs中VGLL4的敲除减弱了BSD对成骨细胞分化的促进作用,这表明VGLL4在BSD的治疗效果中起着至关重要的作用。
结论：BSD通过上调VGLL4水平改善骨质疏松损伤并促进成骨细胞分化，进而拮抗TEAD4介导的RUNX2转录抑制。我们的研究暗示BSD可能是骨质疏松症的治疗剂。
目的：本研究旨在探讨BSD对骨质疏松损伤的保护作用。并探讨BSD是否通过靶向VGLL4调节BMSCs的成骨分化，进而改善PMOP。
方法：在去卵巢（OVX）大鼠和骨髓间充质干细胞（BMSCs）中研究BSD的抗骨质疏松作用。进行Micro-CT成像和HE染色，并测定成骨蛋白RUNX2和成骨相关因子VGLL4的水平。进一步采用免疫共沉淀(Co-IP)来研究BSD对TEAD4和RUNX2之间的相互作用的影响。关键成骨因子1ALP,通过RT-qPCR检测COLl1A1和Osterix的表达。采用Co-IP和邻近连接测定(PLA)来仔细检查BSD对TEAD4和RUNX2相互结合的影响。此外，在BMSCs中进行VGLL4敲低以证实VGLL4在BSD的治疗机制中的作用。
结果：BSD对骨质疏松损伤具有剂量依赖性保护作用，骨体积的改善证明了这一点，骨微结构，和组织形态计量学。此外,BSD处理增加了RUNX2及其下游靶基因包括ALP的水平,COL1A1和Osterix.此外，BSD上调VGLL4表达并减少TEAD4-RUNX2相互作用。在BMSCs实验中,含BSD的血清能促进BMSCs成骨分化,增强成骨相关因子的表达和VGLL4水平。BMSCs中VGLL4的敲除减弱了BSD对成骨细胞分化的促进作用,这表明VGLL4在BSD的治疗效果中起着至关重要的作用。
结论：BSD通过上调VGLL4水平改善骨质疏松损伤并促进成骨细胞分化，进而拮抗TEAD4介导的RUNX2转录抑制。我们的研究暗示BSD可能是骨质疏松症的治疗剂。
