Cao Jiao, Li Yue, Si Mengying, Ma Shaoyang, Li Meng, Shi Anbang, Liu Jin, Li Ang
Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi'an Jiaotong University, Xi'an, Shaanxi, PR China.
Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi'an Jiaotong University, Xi'an, Shaanxi, PR China; Department of Periodontology, College of Stomatology, Xi'an Jiaotong University, Xi'an, Shaanxi, PR China.
Phytomedicine. 2025 Jun;141:156733. doi: 10.1016/j.phymed.2025.156733. Epub 2025 Apr 6.
The osteogenic differentiation of periodontal ligament stem cells (PDLSCs) plays a fundamental role in endogenous bone regeneration during periodontitis treatment, yet achieving consistent differentiation under inflammatory conditions remains clinically challenging. Kaempferol, a phytochemical flavonol, has demonstrated osteoprotective efficacy in osteoporosis and bone repair models. However, whether kaempferol exerts pro-osteogenic effects on PDLSCs within the pathologically complex microenvironment of periodontitis, and through what molecular mechanisms, remains unexplored.
This study aimed to systematically characterize the therapeutic efficacy of kaempferol in restoring osteogenic differentiation of human PDLSCs under inflammatory stress, and promoting bone regeneration in a mice periodontitis model, and elucidate novel molecular targets and downstream mechanisms mediating these regenerative actions.
An in vitro inflammatory microenvironment was established using lipopolysaccharide (LPS)-stimulated human PDLSCs to mimic periodontitis-induced osteogenic impairment. Osteogenic recovery was assessed through alkaline phosphatase (ALP), alizarin red S staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analysis of osteogenesis-related markers (ALP, RUNX2, OSX, OPN). The bioinformatics, network pharmacology and siRNA transfection were performed to identify EphrinB2 as kaempferol's putative cellular target. Downstream PI3K/Akt and p38 MAPK pathway activation was evaluated through phosphoprotein analysis. In vivo validation employed micro-CT quantification of alveolar bone loss and immunohistochemical profiling of pathways key proteins in a mice periodontitis model.
Kaempferol dose-dependently rescued LPS-impaired osteogenic differentiation in human PDLSCs, especially at 10 μM, where kaempferol significantly reversed suppressed ALP activity, mineralized nodule formation, and transcriptional and protein expression of osteogenic markers (ALP, RUNX2, OSX, OPN). Mechanistically, kaempferol upregulated the key target EphrinB2 under inflammatory stress, thereby reactivating the downstream PI3K/Akt and p38 pathways. In periodontitis mice, kaempferol administration (10 mg/kg) significantly promoted the periodontal expression of OPN and EphrinB2, restored the phosphorylation of PI3K, AKT, and P38, attenuating alveolar bone loss by 63.8 % (BV/TV: 72.4 % ± 2.07 vs. 44.2 % ± 3.19 in CON).
Kaempferol could rescue PDLSCs' osteogenic differentiation and mitigates bone loss in periodontitis microenvironments by targeting EphrinB2 to activate PI3K/Akt and P38 pathways. This work underscores kaempferol's potential as a natural therapeutic for reversing pathological bone resorption and promoting periodontal regeneration.
牙周膜干细胞（PDLSCs）的成骨分化在牙周炎治疗期间的内源性骨再生中起着重要作用，但在炎症条件下实现一致的分化在临床上仍然具有挑战性。山奈酚是一种植物化学黄酮醇，已在骨质疏松症和骨修复模型中显示出骨保护功效。然而，山奈酚在牙周炎病理复杂的微环境中是否对PDLSCs发挥促骨生成作用，以及通过何种分子机制发挥作用，仍有待探索。
本研究旨在系统地描述山奈酚在恢复炎症应激下人PDLSCs的成骨分化以及促进小鼠牙周炎模型中的骨再生方面的治疗效果，并阐明介导这些再生作用的新分子靶点和下游机制。
使用脂多糖（LPS）刺激的人PDLSCs建立体外炎症微环境，以模拟牙周炎诱导的成骨损伤。通过碱性磷酸酶（ALP）、茜素红S染色、定量实时聚合酶链反应（qRT-PCR）以及成骨相关标志物（ALP、RUNX2、OSX、OPN）的蛋白质印迹分析来评估成骨恢复情况。进行生物信息学、网络药理学和小干扰RNA（siRNA）转染以确定EphrinB2为山奈酚的假定细胞靶点。通过磷酸化蛋白分析评估下游PI3K/Akt和p38丝裂原活化蛋白激酶（MAPK）途径的激活情况。体内验证采用小鼠牙周炎模型中牙槽骨丢失的显微计算机断层扫描（micro-CT）定量以及途径关键蛋白的免疫组织化学分析。
山奈酚剂量依赖性地挽救了LPS损伤的人PDLSCs的成骨分化，尤其是在10 μM时，山奈酚显著逆转了ALP活性、矿化结节形成以及成骨标志物（ALP、RUNX2、OSX、OPN）的转录和蛋白质表达的抑制。机制上，山奈酚在炎症应激下上调关键靶点EphrinB2，从而重新激活下游PI3K/Akt和p38途径。在牙周炎小鼠中，给予山奈酚（10 mg/kg）显著促进了OPN和EphrinB2的牙周表达，恢复了PI3K、AKT和P38的磷酸化，使牙槽骨丢失减少了63.8%（骨体积分数：72.4%±2.07，而对照组为44.2%±3.19）。
山奈酚可通过靶向EphrinB2激活PI3K/Akt和P38途径，挽救PDLSCs的成骨分化并减轻牙周炎微环境中的骨质流失。这项工作强调了山奈酚作为一种天然疗法在逆转病理性骨吸收和促进牙周再生方面的潜力。
