He Miao, Lin Yangdong
Department of Stomatology, Tianjin First Central Hospital, Nankai District, Tianjin, China.
Biomol Biomed. 2025 Apr 3;25(5):1138-1149. doi: 10.17305/bb.2024.11367.
Human periodontal ligament stem cells (hPDLSCs) play a critical role in the regeneration of periodontal tissue. Forkhead box protein A1 (FOXA1) has been implicated in the inflammatory mechanisms of various diseases. However, the role of FOXA1 in periodontal inflammation and its effect on the osteogenic differentiation of hPDLSCs remains unclear. In this study, healthy tooth root-derived hPDLSCs were isolated, and flow cytometry was used to detect cell surface markers. Western blot and immunofluorescence analyses were performed to assess FOXA1 levels in different tissues. The levels of inflammatory factors were measured using Western blot and ELISA kits. Alkaline phosphatase (ALP) staining, alizarin red S staining, and Western blot were employed to evaluate the impact of FOXA1 silencing on the osteogenic differentiation of hPDLSCs. Finally, the protein levels in the Toll-like receptor 4 (TLR4)/Myeloid differentiation factor-88 (MyD88)/NF-κB pathway were analyzed using Western blot. Results showed that periodontal membrane tissues from patients with periodontitis exhibited a marked increase in FOXA1 levels. Lipopolysaccharide (LPS) treatment significantly upregulated FOXA1 expression in hPDLSCs, elevated inflammatory factor levels, and inhibited osteogenic differentiation. However, silencing FOXA1 mitigated the effects of LPS. Furthermore, LPS treatment activated the TLR4/MyD88/NF-κB pathway, while FOXA1 silencing impeded this activation. Notably, the application of the TLR4 agonist CRX-527 reversed the inhibitory effects of FOXA1 silencing on LPS-induced responses. In summary, silencing FOXA1 reduced cellular inflammation by inhibiting the TLR4/MyD88/NF-κB pathway and alleviated the suppressive effects of LPS on the osteogenic differentiation of hPDLSCs.
人牙周膜干细胞（hPDLSCs）在牙周组织再生中起关键作用。叉头框蛋白A1（FOXA1）与多种疾病的炎症机制有关。然而，FOXA1在牙周炎症中的作用及其对hPDLSCs成骨分化的影响仍不清楚。在本研究中，分离出健康牙根来源的hPDLSCs，并使用流式细胞术检测细胞表面标志物。进行蛋白质免疫印迹和免疫荧光分析以评估不同组织中FOXA1的水平。使用蛋白质免疫印迹和酶联免疫吸附测定试剂盒测量炎症因子水平。采用碱性磷酸酶（ALP）染色、茜素红S染色和蛋白质免疫印迹来评估FOXA1沉默对hPDLSCs成骨分化的影响。最后，使用蛋白质免疫印迹分析Toll样受体4（TLR4）/髓样分化因子88（MyD88）/核因子κB（NF-κB）信号通路中的蛋白质水平。结果显示，牙周炎患者的牙周膜组织中FOXA1水平显著升高。脂多糖（LPS）处理显著上调hPDLSCs中FOXA1的表达，升高炎症因子水平，并抑制成骨分化。然而，沉默FOXA1可减轻LPS的作用。此外，LPS处理激活了TLR4/MyD88/NF-κB信号通路，而FOXA1沉默则阻碍了这种激活。值得注意的是，应用TLR4激动剂CRX-527可逆转FOXA1沉默对LPS诱导反应的抑制作用。总之，沉默FOXA1通过抑制TLR4/MyD88/NF-κB信号通路减少细胞炎症，并减轻LPS对hPDLSCs成骨分化的抑制作用。
