Regulatory Mechanism of Keap1/Nfe2L2 on Osteogenic Differentiation in Periodontitis

Yanfei HUANG; Hongbin YU; Lingyun YIN; Jing LIANG; Changquan LI; Dehong LI; Jinyuan WANG; Qian OUYANG

doi:10.12259/j.issn.2095-610X.S20250704

Volume 46 Issue 7

Jul. 2025

Turn off MathJax

Article Contents

Article Navigation > Journal of Kunming Medical University > 2025 > 46(7): 26-37

Yanfei HUANG, Hongbin YU, Lingyun YIN, Jing LIANG, Changquan LI, Dehong LI, Jinyuan WANG, Qian OUYANG. Regulatory Mechanism of Keap1/Nfe2L2 on Osteogenic Differentiation in Periodontitis[J]. Journal of Kunming Medical University, 2025, 46(7): 26-37. doi: 10.12259/j.issn.2095-610X.S20250704

Citation:

Yanfei HUANG, Hongbin YU, Lingyun YIN, Jing LIANG, Changquan LI, Dehong LI, Jinyuan WANG, Qian OUYANG. Regulatory Mechanism of Keap1/Nfe2L2 on Osteogenic Differentiation in Periodontitis[J]. Journal of Kunming Medical University, 2025, 46(7): 26-37. doi: 10.12259/j.issn.2095-610X.S20250704

Citation:

Yanfei HUANG, Hongbin YU, Lingyun YIN, Jing LIANG, Changquan LI, Dehong LI, Jinyuan WANG, Qian OUYANG. Regulatory Mechanism of Keap1/Nfe2L2 on Osteogenic Differentiation in Periodontitis[J]. Journal of Kunming Medical University, 2025, 46(7): 26-37. doi: 10.12259/j.issn.2095-610X.S20250704

PDF( 4688 KB)

Regulatory Mechanism of Keap1/Nfe2L2 on Osteogenic Differentiation in Periodontitis

doi: 10.12259/j.issn.2095-610X.S20250704

Department of Stomatology，Yan’an Hospital Affiliated to Kunming Medical University，Kunming Yunnan 650051，China

Received Date: 2025-05-05
Publish Date: 2025-07-21

Abstract

Abstract

Objective To explore the regulatory mechanism of NFE2L2/KEAP1 in alveolar bone repair induced by periodontitis. Methods A rat periodontitis model was established and divided into four groups: Control group （n = 6）; Periodontitis model group （n = 6）; Periodontitis + lentivirus empty vector group （n = 6）; Periodontitis + NFE2L2 overexpression plasmid group （n = 6）. Histopathological changes in each group were observed using HE staining. TRAP staining was used to detect osteoclast positivity, while ELISA was employed to measure inflammatory cytokine levels in tissues. Immunofluorescence and qPCR were used to detect NFE2L2 expression, and western blot was used to assess the expression of osteogenic proteins ALPL2, RUNX2, and COL1. Primary periodontal ligament cells （hPDLCs） were cultured, and cells were transfected to overexpress NFE2L2 and KEAP1. The cells were divided into six groups: Normal group; Model group; pcDNA-NC group; pcDNA-NFE2L2 group; pc-NFE2L2 + pcDNA-NC group; pc-NFE2L2 + pcDNA-KEAP1 group. A cellular model was established, and the morphology of primary hPDLCs was observed under a microscope. Cell proliferation was assessed using CCK-8. Osteogenic mineralization was observed using alizarin red staining, and western blot was used to detect osteogenic proteins and autophagy markers. Cell migration was observed using a scratch assay. Results （1） After model induction, redness, swelling of the gums, extensive inflammatory infiltration, and alveolar bone resorption were observed, confirming successful model establishment.Partial tissue recovery occurred after NFE2L2 overexpression via lentivirus. （2） After model induction, osteoclast positivity increased, confirming successful model establishment. Overexpression of NFE2L2 reduced osteoclast positivity （P < 0.001）. （3） After model induction, levels of IL-1β, IL-10, and TNF-α were significantly higher than in the normal group （P < 0.05）, confirming successful model establishment. Transfection with NFE2L2 lentivirus reduced inflammatory cytokine levels （P < 0.0001）. After model induction, osteogenic protein expression decreased compared to the normal group, but overexpression of NFE2L2 increased osteogenic protein expression （P < 0.05）. （5） LPS treatment significantly reduced cell viability, while NFE2L2 overexpression enhanced it （P < 0.0001）. （6） LPS treatment reduced calcified nodules, while NFE2L2 overexpression increased them. Addition of pcDNA-KEAP1 reduced mineralized nodules. （7） LPS treatment decreased osteogenic protein expression, while NFE2L2 overexpression increased it. However, addition of pcDNA-KEAP1 reduced osteogenic protein expression （P < 0.05）. （8） LPS treatment reduced cell migration, whereas NFE2L2 overexpression enhanced it （P < 0.0001）. （9） Expression of autophagy markers decreased after LPS treatment, but increased after transfection with NFE2L2 plasmid. However, addition of pcDNA-KEAP1 reduced the expression of autophagy markers （P < 0.05）. Conclusion This study identified the regulatory role of NFE2L2/KEAP1 in periodontitis, providing a scientific basis for the treatment of periodontitis.
- Periodontitis,
- KEAP1/NFE2L2,
- Autophagy,
- Osteoblasts

FullText(HTML)

References(32)

References

[1]	Laky M,Arslan M,Zhu X,et al. Quercetin in the prevention of induced periodontal disease in animal models:A systematic review and meta-analysis[J]. Nutrients,2024,16(5):735. doi: 10.3390/nu16050735
[2]	Chen M X,Zhong Y J,Dong Q Q,et al. Global,regional,and national burden of severe periodontitis,1990-2019:An analysis of the Global Burden of Disease Study 2019[J]. J Clin Periodontol,2021,48(9):1165-1188. doi: 10.1111/jcpe.13506
[3]	Isola G. The impact of diet,nutrition and nutraceuticals on oral and periodontal health[J]. Nutrients,2020,12(9):E2724. doi: 10.3390/nu12092724
[4]	周婷,徐屹,丁一,赵寰. 慢性牙周炎龈下菌斑中五种牙周可疑致病微生物的分布[J]. 华西口腔医学杂志,2007,25(5):4.
[5]	Stefanescu A,Sufaru I G,Martu C,et al. Impact of infrabony defects treatment on periodontal markers and glycated hemoglobin using platelet-rich fibrin,guided tissue regeneration,and access-flap surgery[J]. Medicina (Kaunas),2024,60(11):1769. doi: 10.3390/medicina60111769
[6]	Shiheido-Watanabe Y,Maejima Y,Nakagama S,et al. Porphyromonas gingivalis,a periodontal pathogen,impairs post-infarcted myocardium by inhibiting autophagosome-lysosome fusion[J]. Int J Oral Sci,2023,15(1):42. doi: 10.1038/s41368-023-00251-2
[7]	Chapple I L,Genco R,working group 2 of the joint EFP/AAP workshop. Diabetes and periodontal diseases:Consensus report of the Joint EFP/AAP Workshop on Periodontitis and Systemic Diseases[J]. J Periodontol,2013,84(4 suppl):S106-S112.
[8]	Morandini A C,Ramos-Junior E S. Mitochondrial function in oral health and disease[J]. J Immunol Meth,2024,532:113729. doi: 10.1016/j.jim.2024.113729
[9]	Hashim N T,Fathima S,Hisham N M,et al. Exploring salivary alpha-amylase as a biomarker in periodontitis:A comparative analysis of disease stages and clinical correlations[J]. Curr Issues Mol Biol,2024,46(11):12230-12243. doi: 10.3390/cimb46110726
[10]	İnan S,Barış E. The role of autophagy in odontogenesis,dental implant surgery,periapical and periodontal diseases[J]. J Cell Mol Med,2024,28(8):e18297. doi: 10.1111/jcmm.18297
[11]	Yang J W,Zhu L X,Yuan G H,et al. Autophagy appears during the development of the mouse lower first molar[J]. Histochem Cell Biol,2013,139(1):109-118. doi: 10.1007/s00418-012-1016-2
[12]	Pantovic A,Krstic A,Janjetovic K,et al. Coordinated time-dependent modulation of AMPK/Akt/mTOR signaling and autophagy controls osteogenic differentiation of human mesenchymal stem cells[J]. Bone,2013,52(1):524-531. doi: 10.1016/j.bone.2012.10.024
[13]	Mysak J,Podzimek S,Sommerova P,et al. Porphyromonas gingivalis:Major periodontopathic pathogen overview[J]. J Immunol Res,2014,2014:476068.
[14]	Wan X,He H,Jialing L,et al. Hippo-YAP signaling pathway regulates autophagy of human periodontal ligament cells under cyclic tensile stress[J]. Hua Xi Kou Qiang Yi Xue Za Zhi,2023,41(3):260-268.
[15]	罗敬一,程波. 基于转录组学探讨氧化应激相关基因在牙周炎中的作用机制[J]. 临床口腔医学杂志,2023,1(1):1.
[16]	Qi Y,Luan Y,Wang L. Low concentrations of tumor necrosis factor-alpha promote human periodontal ligament stem cells osteogenic differentiation by activation of autophagy via inhibition of AKT/mTOR pathway[J]. Mol Biol Rep,2023,50(4):3329-3339. doi: 10.1007/s11033-022-08173-8
[17]	Uribe-Carretero E,Martinez-Chacón G,Yakhine-Diop S M S,et al. Loss of KEAP1 causes an accumulation of nondegradative organelles[J]. Antioxidants (Basel),2022,11(7):1398. doi: 10.3390/antiox11071398
[18]	Liu Q,Fan W,He Y,et al. Effects of melatonin on the proliferation and differentiation of human dental pulp cells[J]. Arch Oral Biol,2017,83:33-39. doi: 10.1016/j.archoralbio.2017.06.034
[19]	Wang Q,Shi W,Lin S,et al. FOXO1 regulates osteogenic differentiation of periodontal ligament stem cells through the METTL3 signaling pathway[J]. J Orthop Surg Res,2023,18(1):637. doi: 10.1186/s13018-023-04120-w
[20]	杨宇,孙敬涛,王陈保. 牙龈卟啉单胞菌感染对人牙周膜细胞的增殖与成骨分化及炎性因子的影响[J]. 中华医院感染学杂志,2018,28(12):5.
[21]	陈斌,徐蓉蓉,张家鼎,闫福华. 重度牙周炎患牙的保存治疗[J]. 国际口腔医学杂志,2020,47(2):6.
[22]	李岩涛,贺兰,李洁,等. 牙周炎龈组织肿瘤坏死因子α的含量及其与牙周炎的关系[J]. 口腔医学,2002,22(1):3. doi: 10.3969/j.issn.1003-9872.2002.01.002
[23]	邓行华,牟云,蒋峻嵩. 盐酸米诺环素软膏联合牙龈下刮治,根面平整术治疗牙周炎并牙龈出血效果观察[J]. 解放军医药杂志,2022,1(2):34.
[24]	Tonetti M S,Greenwell H,Kornman K S. Staging and grading of periodontitis:Framework and proposal of a new classification and case definition[J]. J Periodontol,2018,89(Suppl 1):S159-S172.
[25]	Liu S,Ge J,Chu Y,et al. Identification of hub cuproptosis related genes and immune cell infiltration characteristics in periodontitis[J]. Front Immunol,2023,14:1164667. doi: 10.3389/fimmu.2023.1164667
[26]	Sima C,Aboodi G M,Lakschevitz F S,et al. Nuclear factor erythroid 2-related factor 2 down-regulation in oral neutrophils is associated with periodontal oxidative damage and severe chronic periodontitis[J]. Am J Pathol,2016,186(6):1417-1426. doi: 10.1016/j.ajpath.2016.01.013
[27]	Eid F E,ElHefnawi M,Heath L S. DeNovo:Virus-host sequence-based protein-protein interaction prediction[J]. Bioinformatics,2016,32(8):1144-1150. doi: 10.1093/bioinformatics/btv737
[28]	Chen J,Hu C,Lu X,et al. ALDH2 alleviates inflammation and facilitates osteogenic differentiation of periodontal ligament stem cells in periodontitis by blocking ferroptosis via activating Nrf2[J]. Funct Integr Genomics,2024,24(5):184. doi: 10.1007/s10142-024-01465-1
[29]	Lu H,Zheng Y,Wang D. ATF3 affects osteogenic differentiation in inflammatory hPDLSCs by mediating ferroptosis via regulating the Nrf2/HO-1 signaling pathway[J]. Tissue Cell,2024,89:102447. doi: 10.1016/j.tice.2024.102447
[30]	Tossetta G,Fantone S,Togni L,et al. Modulation of NRF2/KEAP1 signaling by phytotherapeutics in periodontitis[J]. Antioxidants (Basel),2024,13(10):1270. doi: 10.3390/antiox13101270
[31]	Mukherjee R,Bhattacharya A,Mello-Vieira J,et al. Serine ubiquitination of SQSTM1 regulates NFE2L2-dependent redox homeostasis[J]. Autophagy,2025,21(2):407-423. doi: 10.1080/15548627.2024.2404375
[32]	Gou H,Chen X,Zhu X,et al. Sequestered SQSTM1/p62 crosstalk with Keap1/NRF2 axis in hPDLCs promotes oxidative stress injury induced by periodontitis[J]. Free Radic Biol Med,2022,190:62-74. doi: 10.1016/j.freeradbiomed.2022.08.001

Relative Articles(20)

Relative Articles

[1]	Yuan FANG, Kunlun WU, Shitao ZHANG, Yang FENG. Correlation between Platelet Autophagy Related Factors and Neurological Damage and Prognosis in Patients with Aneurysmal Subarachnoid Hemorrhage. Journal of Kunming Medical University, 2025, 46(5): 118-125. doi: 10.12259/j.issn.2095-610X.S20250514
[2]	Xiaoxiao FAN, Xuan ZENG, Pingping ZHOU, Xi LIU, Feifei ZHU, Yanji LUO, Yi WU. Relationship between the Platelet Autophagy-related Factor Expression and Peritoneal Metastasis of Gastric Cancer. Journal of Kunming Medical University, 2025, 46(7): 125-130. doi: 10.12259/j.issn.2095-610X.S20250715
[3]	Yilin ZHU, Xiao PENG, Guifu ZHANG, Huinan LONG. Effects of Autophagy on Chondrocyte Apoptosis in Osteoarthritis: An Investigation Based on lncRNA/Hedgehog Signaling Pathway Expression. Journal of Kunming Medical University, 2025, 46(6): 38-45. doi: 10.12259/j.issn.2095-610X.S20250605
[4]	Ximeng SONG, Shilong YUAN, Zi'ang LU, Maotong XU. Exploring the Molecular Mechanism of Ligustrazine in Spinal Cord Injury Repair Based on Macrophage Autophagy. Journal of Kunming Medical University, 2025, 46(7): 38-45. doi: 10.12259/j.issn.2095-610X.S20250705
[5]	Lili YANG, Ting ZHOU, Xiaobin REN, Jianhua WU, Yi PENG, Yanqing SHUI. Prospective Clinical Study of Combined Treatment of Periodontal Orthodontics for Chronic Periodontitis. Journal of Kunming Medical University, 2024, 45(3): 54-58. doi: 10.12259/j.issn.2095-610X.S20240308
[6]	Xuesha ZHANG, Jie ZHOU, Cailin LI, Dingyu WU, Fang PENG. Effects of Periplaneta americana Extract on Autophagy and Invasive Metastasis of Subcutaneous Transplanted Hepatocellular Carcinoma in Nude Mice. Journal of Kunming Medical University, 2023, 44(10): 39-46. doi: 10.12259/j.issn.2095-610X.S20231018
[7]	Chenchen LIANG, Jianpeng GAO. MSCs Promote Autophagy to Alleviate Liver Injury in NASH by Regulating AMPK/mTOR Axis. Journal of Kunming Medical University, 2023, 44(7): 162-167. doi: 10.12259/j.issn.2095-610X.S20230728
[8]	Shi CHEN, Shi FU, Zhiyong TAN, Jiansong WANG, Haifeng WANG. Research Progress of Autophagy in the Development and Treatment of Bladder Cancer. Journal of Kunming Medical University, 2023, 44(5): 168-174. doi: 10.12259/j.issn.2095-610X.S20230503
[9]	Shiyuan YANG, Hao ZHANG, Tuqiang HU. Effect of Pristimerin on Proliferation of Oral Squamous Cell Carcinoma CAL-27 by Regulating Autophagy. Journal of Kunming Medical University, 2023, 44(10): 92-99. doi: 10.12259/j.issn.2095-610X.S20231024
[10]	Yixuan CHEN, Lin WANG, Xiuhong XIA, Zaikun PENG, Yi DING, Runjiao SHI, Xuefen LEI. Research Progress on the Mechanisms of Autophagy in Hepatocellular Carcinoma. Journal of Kunming Medical University, 2023, 44(4): 159-164. doi: 10.12259/j.issn.2095-610X.S20230416
[11]	Ziwei ZHANG, Jialin ZHENG, Xiaoyu XU, Hong WANG. Metformin Inhibits high Glucose-induced Vascular Smooth Muscle Cells Proliferation by Promoting Autophagy via AMPK/PPAR-γ. Journal of Kunming Medical University, 2023, 44(10): 60-66. doi: 10.12259/j.issn.2095-610X.S20231026
[12]	Le ZHAN, Ruiyu MA, Ni WAN, Liwen MO, Huiying CHEN, Li ZHANG. Effects of Puerarin on Th17/Treg Cell Immune Homeostasis and the Expression of Related Transcription Factors in Rats with Periodontitis. Journal of Kunming Medical University, 2022, 43(11): 36-43. doi: 10.12259/j.issn.2095-610X.S20221129
[13]	Yong-jun LIU, Yin WANG, Mei YANG, E-gao YIN, Ting LI, Zhao-xing DONG. The Effect of Hydrogen-Regulated Autophagy on Pulmonary Fibrosis in Paraquat Poisoned Rats. Journal of Kunming Medical University, 2021, 42(9): 1-6. doi: 10.12259/j.issn.2095-610X.S20210918
[14]	Yun LIU, Hong-bing HE. The Research Progress on the Effect of Prostaglandin E2 on Bone Remodeling. Journal of Kunming Medical University, 2021, 42(9): 149-155. doi: 10.12259/j.issn.2095-610X.S20210916
[15]	Xu ZHENG, Chen XIE, Rao FU, Yu-zhi WU, Zhu-ling GUO. A Survey on the Cognition of the Relationship between Periodontitis and Diabetes Mellitus in Type 2 Diabetes Patients and Endocrinologists. Journal of Kunming Medical University, 2021, 42(3): 89-93. doi: 10.12259/j.issn.2095-610X.S20210323
[16]	Shen Bao Yu , Yang Gen Meng , Li Yuan Yuan , Liu Liu , Huang Jian , Zeng Xiao Feng , Li Li Hua . . Journal of Kunming Medical University, 2020, 41(01): 163-167.
[17]	Wang Xiang Yun . . Journal of Kunming Medical University,
[18]	Ni Tao . . Journal of Kunming Medical University,
[19]	Cao Xiao Bo . . Journal of Kunming Medical University,
[20]	Zhang Yi Fan . . Journal of Kunming Medical University,