Tan Lihua, Miao Zhimin, Zhao Yuxin, Liang Yongkai, Xu Nan, Chen Xin, Tu Yanbei, He Chengwei
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR 999078, China.
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR 999078, China; School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu 212013, China.
J Adv Res. 2024 Dec 9. doi: 10.1016/j.jare.2024.12.009.
Osteoporosis is an osteolytic disorder resulting from an inequilibrium between osteoblast-mediated osteogenesis and osteoclast-driven bone absorption. Safe and effective approaches for osteoporosis management are still highly demanded.
This study aimed to examine the osteoprotective effect and the mechanisms of phaseol (PHA) in vitro and in vivo.
Virtual screening identified the potential inhibitors of transforming growth factor-beta-activated kinase 1 (TAK1) from coumestans. The interaction between PHA and TAK1 was investigated by molecular simulation, pronase and thermal resistance assays. The maturation and function of osteoclasts were determined using tartrate-resistant acid phosphatase staining, bone absorption and F-actin ring formation assays. The differentiation and calcification of osteoblasts were assessed by alkaline phosphatase staining and Alizarin Red S staining. The activity of related targets and pathways were detected using immunoblotting, immunofluorescence and co-immunoprecipitation assays. The in vivo osteoprotective effect of PHA was evaluated using a lipopolysaccharide (LPS)-induced mouse osteoporosis model.
Firstly, we confirmed that TAK1 was essential in controlling bone remodeling by regulating osteogenesis and osteoclastogenesis. Moreover, PHA, a coumestan compound predominantly present in leguminous plants, was identified as a potent TAK1 inhibitor through virtual and real experiments. Subsequently, PHA was observed to enhance osteoblast differentiation and calcification, while suppress osteoclast maturation and bone resorptive function in vitro. Mechanistically, PHA remarkably inhibited the TRAF6-TAK1 complex formation, and inhibited the activation of TAK1, MAPK and NF-κB pathways by targeting TAK1. In the in vivo study, PHA strongly attenuated bone loss, inflammatory responses, and osteoclast over-activation in lipopolysaccharide-induced osteoporosis mice.
PHA had a dual-functional regulatory impact on osteogenesis and osteoclastogenesis by targeting TAK1, suppressing TRAF6-TAK1 complex generation, and modulating its associated signaling pathways, ultimately leading to mitigating osteoporosis. This study offered compelling evidence in favor of using PHA for preventing and managing osteoporosis as both a bone anabolic and anti-resorptive agent.
骨质疏松症是一种溶骨性疾病，由成骨细胞介导的骨生成与破骨细胞驱动的骨吸收之间的不平衡所致。目前仍迫切需要安全有效的骨质疏松症治疗方法。
本研究旨在探讨菜豆素（PHA）在体外和体内的骨保护作用及其机制。
通过虚拟筛选从香豆雌酚中鉴定出潜在的转化生长因子-β激活激酶1（TAK1）抑制剂。采用分子模拟、链霉蛋白酶和耐热性试验研究PHA与TAK1之间的相互作用。使用抗酒石酸酸性磷酸酶染色、骨吸收和F-肌动蛋白环形成试验测定破骨细胞的成熟和功能。通过碱性磷酸酶染色和茜素红S染色评估成骨细胞的分化和钙化。使用免疫印迹、免疫荧光和免疫共沉淀试验检测相关靶点和信号通路的活性。采用脂多糖（LPS）诱导的小鼠骨质疏松模型评估PHA的体内骨保护作用。
首先，我们证实TAK1通过调节骨生成和破骨细胞生成在控制骨重塑中起关键作用。此外，通过虚拟和实际实验，PHA（一种主要存在于豆科植物中的香豆雌酚化合物）被鉴定为一种有效的TAK1抑制剂。随后，观察到PHA在体外可增强成骨细胞分化和钙化，同时抑制破骨细胞成熟和骨吸收功能。机制上，PHA显著抑制TRAF6-TAK1复合物的形成，并通过靶向TAK1抑制TAK1、MAPK和NF-κB信号通路的激活。在体内研究中，PHA强烈减轻脂多糖诱导的骨质疏松小鼠的骨质流失、炎症反应和破骨细胞过度激活。
PHA通过靶向TAK1、抑制TRAF6-TAK1复合物的产生并调节其相关信号通路，对骨生成和破骨细胞生成具有双重调节作用，最终减轻骨质疏松症。本研究提供了有力证据，支持将PHA作为一种骨合成代谢和抗吸收剂用于预防和治疗骨质疏松症。
