Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon, Gangwon-Do 24341, Republic of Korea.
Kangwon Institute of Inclusive Technology, Kangwon National University, Chuncheon, Gangwon-Do 24341, Republic of Korea.
Phytomedicine. 2022 Oct;105:154378. doi: 10.1016/j.phymed.2022.154378. Epub 2022 Aug 3.
Elevated activity of osteoclasts (OCs) is linked to osteolytic bone diseases, such as osteoporosis and rheumatoid arthritis. Developing natural anti-osteoclastogenic compounds with greater efficacy and fewer adverse effects is crucial for preventing or treating osteolytic bone diseases. N-triterpene cycloartane saponins (NTCSs) are rarely found in nature, and their inhibitory effects on OC differentiation in vitro and in vivo have not yet been explored.
This study was aimed to investigate the effect of mussaendoside O, an NTCS isolated from Mussaenda pubescens, on RANKL-induced OC differentiation and its underlying mechanism in vitro, and lipopolysaccharide (LPS)-induced bone resorption in a mouse model.
The content of mussaendoside O in methanol extract of M. pubescens was determined by HPLC. The inhibitory effects of mussaendoside O on RANKL-induced OC formation were assessed using TRAP staining, western blotting, immunofluorescence staining, and real-time qPCR. Meanwhile, the effects of mussaendoside O on LPS-induced inflammatory responses were assessed using a Griess reagent and qPCR. The effects of mussaendoside O on LPS-induced bone resorption in a mouse model were evaluated using micro-CT and immunohistochemical staining.
Mussaendoside O inhibited RANKL-induced TRAP-positive multinucleated OC formation in a concentration-dependent manner without affecting cell viability. However, mussaendoside O did not inhibit LPS-induced mRNA expression of COX-2, iNOS, and TNF-α. Mice orally administrated with mussaendoside O exhibited significant protection from LPS-induced bone resorption and OC formation. At the molecular level, mussaendoside O suppressed RANKL-activated phosphorylation of p38 MAPK and JNK, as well as c-Fos expression. In addition, mussaendoside O suppressed RANKL-induced NFATc1 activation and the expression of its target genes, including OSCAR, DC-STAMP, CtsK, and TRAP.
Mussaendoside O attenuates OC differentiation in vitro and LPS-induced bone resorption in a mouse model by inhibiting the RANKL-activated c-Fos/NFATc1 signaling pathways. Therefore, mussaendoside O may be a valuable lead compound for preventing or treating of osteolytic bone diseases.
破骨细胞（OCs）活性升高与溶骨性骨疾病有关，如骨质疏松症和类风湿性关节炎。开发具有更高疗效和更少不良反应的天然抗破骨细胞生成化合物对于预防或治疗溶骨性骨疾病至关重要。N-三萜环阿屯烷皂苷（NTCS）在自然界中很少见，其体外和体内抑制 OC 分化的作用尚未得到探索。
本研究旨在探讨从钝叶决明中分离得到的 NTCS 木犀草苷 O 对 RANKL 诱导的 OC 分化的影响及其在体外和 LPS 诱导的小鼠模型中骨吸收的作用机制。
采用 HPLC 法测定钝叶决明甲醇提取物中木犀草苷 O 的含量。采用 TRAP 染色、western blot、免疫荧光染色和实时 qPCR 评估木犀草苷 O 对 RANKL 诱导的 OC 形成的抑制作用。同时，采用 Griess 试剂和 qPCR 评估木犀草苷 O 对 LPS 诱导的炎症反应的影响。采用 micro-CT 和免疫组织化学染色评估木犀草苷 O 对 LPS 诱导的小鼠模型中骨吸收的影响。
木犀草苷 O 呈浓度依赖性抑制 RANKL 诱导的 TRAP 阳性多核 OC 形成，而不影响细胞活力。然而，木犀草苷 O 并不抑制 LPS 诱导的 COX-2、iNOS 和 TNF-α 的 mRNA 表达。经口给予木犀草苷 O 的小鼠对 LPS 诱导的骨吸收和 OC 形成具有显著的保护作用。在分子水平上，木犀草苷 O 抑制了 RANKL 激活的 p38 MAPK 和 JNK 的磷酸化以及 c-Fos 的表达。此外，木犀草苷 O 抑制了 RANKL 诱导的 NFATc1 激活及其靶基因 OSCAR、DC-STAMP、CtsK 和 TRAP 的表达。
木犀草苷 O 通过抑制 RANKL 激活的 c-Fos/NFATc1 信号通路，减轻体外 OC 分化和 LPS 诱导的小鼠模型中的骨吸收。因此，木犀草苷 O 可能是预防或治疗溶骨性骨疾病的有价值的先导化合物。
