Zhao Qiangqiang, Chen Xingling, Mai Bin, Che Feihong, Zhang Zhen, Kang Pan, Hou Chengyu, Lu Lu, Xu Liangliang
Lingnan Medical Research Center, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China.
Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou, China.
Front Pharmacol. 2024 Dec 16;15:1527494. doi: 10.3389/fphar.2024.1527494. eCollection 2024.
Osteoporosis is a pervasive bone metabolic disorder characterized by the progressive degeneration of bone microstructure. Osteoclasts are playing a pivotal role in bone remodeling and resorption. Consequently, modulating osteoclast activity, particularly curbing their overactivation, has become a crucial strategy in clinical treatments. Stigmasterol (STG), a plant-derived phytosterol, has shown promise in inhibiting osteoclastic activity, although its precise biological mechanisms require further scientific investigation. Therefore, this study aims to explore the potential mechanisms by which STG inhibits osteoclasts and to further assess its impact on osteoporosis by establishing an Ovariectomy (OVX) model.
Initially, osteoclast differentiation was induced using RANKL (Receptor Activator of Nuclear Factor Kappa-B Ligand) on RAW 264.7 cells, followed by TRAP staining and F-actin banding to observe the effects of various concentrations of STG during osteoclast differentiation. The osteoclast-specific gene and protein expression changes were further analyzed using Real-Time PCR (qPCR) and Western blot, exploring the RANKL-mediated NF-κB and MAPK signaling pathways. An OVX model was established to examine changes in bone mass through Micro-CT and Hematoxylin and eosin (H&E) staining, and to assess osteoclast formation and characteristic protein expression through TRAP staining and Immunohistochemistry staining.
experiments revealed that STG significantly inhibited osteoclast activity, as evidenced by reductions in osteoclast numbers and spreading areas, and a marked suppression of F-actin formation. On the molecular level, this compound effectively downregulated key osteoclast markers such as NFATc1, Acp5, c-Fos, and ΜMP9 in both gene and protein expressions. Western blot analysis showed that STG notably inhibited the phosphorylation of the p65 subunit in the NF-κB pathway, thus affecting the pathway's activity. Further validation through OVX model indicated significant protective effects of STG against bone loss, as demonstrated by Micro-CT. Histopathological staining confirmed STG's efficacy in reducing bone surface area and volume loss. Additionally, TRAP staining showed significant reductions in osteoclast number and surface area in the STG group compared to the OVX group, underscoring STG's potential therapeutic role in bone metabolism regulation.
The findings reveal that STG effectively inhibits the phosphorylation of the p65 protein in the NF-κB pathway, and influences the MAPK signaling pathway, thereby reducing osteoclast formation and preserving bone mass. These mechanisms provide a crucial molecular basis for its potential therapeutic application in treating osteoporosis.
骨质疏松症是一种普遍存在的骨代谢紊乱疾病,其特征是骨微结构逐渐退化。破骨细胞在骨重塑和吸收过程中起关键作用。因此,调节破骨细胞活性,特别是抑制其过度活化,已成为临床治疗的关键策略。豆甾醇(STG)是一种植物来源的植物甾醇,已显示出抑制破骨细胞活性的潜力,尽管其确切的生物学机制需要进一步的科学研究。因此,本研究旨在探讨STG抑制破骨细胞的潜在机制,并通过建立卵巢切除(OVX)模型进一步评估其对骨质疏松症的影响。
首先,在RAW 264.7细胞上使用核因子κB受体活化因子配体(RANKL)诱导破骨细胞分化,然后进行抗酒石酸酸性磷酸酶(TRAP)染色和F-肌动蛋白带纹观察不同浓度STG在破骨细胞分化过程中的作用。使用实时定量聚合酶链反应(qPCR)和蛋白质免疫印迹法进一步分析破骨细胞特异性基因和蛋白质表达变化,探索RANKL介导的核因子κB(NF-κB)和丝裂原活化蛋白激酶(MAPK)信号通路。建立OVX模型,通过显微计算机断层扫描(Micro-CT)和苏木精-伊红(H&E)染色检查骨量变化,并通过TRAP染色和免疫组织化学染色评估破骨细胞形成和特征性蛋白表达。
实验表明,STG显著抑制破骨细胞活性,表现为破骨细胞数量和铺展面积减少,以及F-肌动蛋白形成明显受抑制。在分子水平上,该化合物在基因和蛋白质表达上均有效下调关键破骨细胞标志物,如活化T细胞核因子1(NFATc1)、抗酒石酸酸性磷酸酶5(Acp5)、原癌基因c-Fos和基质金属蛋白酶9(ΜMP9)。蛋白质免疫印迹分析表明,STG显著抑制NF-κB途径中p65亚基的磷酸化,从而影响该途径的活性。通过OVX模型进一步验证表明,Micro-CT显示STG对骨质流失具有显著的保护作用。组织病理学染色证实STG在减少骨表面积和体积损失方面的有效性。此外,TRAP染色显示,与OVX组相比,STG组破骨细胞数量和表面积显著减少,突出了STG在骨代谢调节中的潜在治疗作用。
研究结果表明,STG有效抑制NF-κB途径中p65蛋白的磷酸化,并影响MAPK信号通路,从而减少破骨细胞形成并维持骨量。这些机制为其在治疗骨质疏松症中的潜在治疗应用提供了关键的分子基础。