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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2025 Vol.26 No.9 P.881-896
NRF2 nuclear translocation and interaction with DUSP1 regulate the osteogenic differentiation of murine mandibular osteoblasts stimulated withPorphyromonas gingivalis lipopolysaccharide
Abstract: BackgroundPeriodontitis is characterized by alveolar bone resorption, aggravated by osteoblast dysfunction, and associated with intracellular oxidative stress linked to the nuclear factor erythroid 2-related factor 2 (NRF2) level. We evaluated the molecular mechanism of periodontitis onset and development and the role of NRF2 in osteogenic differentiation.
MethodsPrimary murine mandibular osteoblasts were extracted and exposed toPorphyromonas gingivalis lipopolysaccharide (Pg-LPS) or other stimuli. Reactive oxygen species (ROS) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining were used to detect intracellular oxidative stress. Alkaline phosphatase staining and alizarin red S staining were used to detect the osteogenic differentiation of osteoblasts. Immunofluorescence and western blotting were used to determine the changes in the mitogen-activated protein kinase (MAPK) pathway and related molecule activities. Immunofluorescence colocalization and co-immunoprecipitation were performed to examine the nuclear translocation of NRF2 and its interaction with dual-specific phosphatase 1 (DUSP1) in cells.
ResultsLigated tissue samples showed higher alveolar bone resorption rate and lower NRF2 level than healthy periodontal tissue samples.Pg-LPS increased intracellular oxidative stress levels and inhibited osteogenic differentiation, whereas changes in NRF2 expression were correlated with changes in the oxidative stress and osteogenesis rate. NRF2 promoted the dephosphorylation of the MAPK pathway by nuclear translocation and the upregulation of DUSP1 expression, thus enhancing the osteogenic differentiation capacity of mandibular osteoblasts. The interaction between NRF2 and DUSP1 was observed.
ConclusionsNRF2 and its nuclear translocation can regulate the osteogenic differentiation of mandibular osteoblasts underPg-LPS conditions by interacting with DUSP1 in a process linked to the MAPK pathway. These findings form the basis of periodontitis treatment.
Key words: Periodontitis; Nuclear factor erythroid 2-related factor 2 (NRF2); Dual-specific phosphatase 1 (DUSP1); Mitogen-activated protein kinase (MAPK); Oxidative stress; Osteogenesis
1浙江大学医学院附属第二医院牙周病专科，中国杭州市，310009
2浙江大学医学院附属第二医院肿瘤研究所，中国杭州市，310009
摘要：本研究旨在探讨在牙龈卟啉单胞菌脂多糖（Pg-LPS）作用下，核因子E2相关因子2（NRF2）与双特异性磷酸酶1（DUSP1）的互作对成骨细胞分化的影响及其潜在分子机制。我们通过构建小鼠实验性牙周炎模型，采用显微计算机断层扫描和免疫组化染色观察小鼠牙槽骨吸收及牙周组织区域内NRF2表达情况，同时采用免疫组化染色法检测牙周健康者和牙周炎患者牙龈及牙槽骨组织中NRF2含量变化。本研究通过Pg-LPS刺激小鼠来源的下颌骨成骨细胞，采用活性氧（ROS）染色和线粒体膜电位检测试剂盒检测细胞氧化应激水平；通过碱性磷酸酶（ALP）染色和茜素红S染色等评估细胞成骨分化能力；采用免疫荧光和蛋白质免疫印迹测定NRF2、DUSP1、成骨分化和丝裂原活化蛋白激酶（MAPK）通路相关基因和蛋白变化；利用免疫荧光染色和免疫共沉淀法检测成骨细胞中NRF2核转位功能及其与DUSP1的相互作用。研究结果发现，与健康组相比，牙周炎组小鼠发生严重的牙槽骨吸收，并在牙周炎小鼠局部牙周组织区域内观察到NRF2低表达。同时，在牙周炎患者牙龈组织与牙槽骨组织中也同样检测到NRF2表达水平的降低。体外实验发现，Pg-LPS呈浓度依赖性增加细胞内ROS含量，并降低细胞线粒体膜电位。Pg-LPS也可抑制成骨分化相关基因蛋白Runt相关转录因子2（RUNX2）、ALP和骨钙素（OCN）的表达，并减少ALP阳性细胞数及矿化结节数量。同时本研究发现，Pg-LPS刺激可显著降低NRF2表达与核转位水平。当加入NRF2抑制剂时，细胞氧化应激活动增强，成骨分化受到抑制；而NRF2激活剂可减弱细胞氧化应激活动，并促进细胞成骨分化。在NRF2抑制剂或小干扰核糖核酸（siRNA）的作用下，DUSP1表达量降低，MAPK通路磷酸化水平上升；加入NRF2激活剂或过表达NRF2基因时，则结果相反。此外，NRF2与DUSP1间可能存在蛋白互作。综上，NRF2通过核转位功能与DUSP1互作，并经MAPK通路调控Pg-LPS条件下小鼠下颌骨成骨细胞的成骨分化，这为牙周炎治疗提供了新思路。
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DOI:
10.1631/jzus.B2400203
CLC number:
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On-line Access:
2025-06-23
Received:
2024-04-22
Revision Accepted:
2024-08-10
Crosschecked:
2025-09-23