Regulatory Mechanism of Keap1/Nfe2L2 on Osteogenic Differentiation in Periodontitis
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摘要:
目的 探讨NFE2L2/KEAP1在牙周炎导致的牙槽骨流失后修复中的调控机制。 方法 建立大鼠牙周炎模型，分为对照组（n = 6）；牙周炎模型组（n = 6）；牙周炎+慢病毒空载组（n = 6）；牙周炎+NFE2L2过表达质粒组（n = 6）。HE观察每组小鼠病理变化，通过TRAP染色检测破骨细胞阳性情况，通过ELISA检测组织中的炎症因子水平，通过免疫荧光和qPCR检测NFE2L2的表达，通过western blot检测成骨蛋白ALPL2， RUNX2以及COL1的表达。培养原代牙周韧带细胞（hPDLCs），通过细胞转染过表达NFE2L2 及KEAP1。细胞分为六组：正常组；模型组；pcDNA-NC组；pcDNA-NFE2L2组；pc-NFE2L2+pcDNA-NC组；pc-NFE2L2+pcDNA-KEAP1组。建立细胞模型，通过显微镜观察原代细胞牙周韧带细胞（hPDLCs）的形态，通过CKK-8观察细胞的增殖，通过茜素红染色法观察成骨细胞矿化，通过Western Blot检测成骨蛋白以及自噬标志物的表达，通过细胞划痕实验观察细胞迁移。 结果 （1）经模型诱导后，牙龈发红、肿胀，出现大量炎性浸润，牙槽骨吸收馅窝，证明模型建立成功，在经过慢病毒过表达NFE2L2后，组织部分恢复；（2）经模型诱导后破骨细胞阳性率上升，证明模型建立成功，过表达NFE2L2减少了破骨细胞的阳性率（P < 0.001）；（3）经模型诱导后，IL-1β，IL-10，及 Tnf-α的浓度相较于正常组均上升（P < 0.05），证明模型建立成功，转染NFE2L2慢病毒之后，炎症因子的浓度减少（P < 0.0001 ）；（4）经模型诱导后，成骨蛋白的表达相较于正常组有所下降过表达NFE2L2之后促进了成骨相关蛋白的表达（P < 0.05）；（5）LPS处理后细胞活力明显下降，过表达NFE2L2增加了细胞的活力（P <0.0001 ）；（6）LPS处理后钙化结节减少，过表达NFE2L2后，钙化结节增加，加入pcDNA-KEAP1后，矿化结节减少；（7）LPS处理后成骨蛋白的表达下降，转染NFE2L2过表达质粒后，成骨相关蛋白的表达增加，但加入pcDNA-KEAP1后，成骨相关蛋白的表达减少（P < 0.05）；（8）LPS处理后细胞迁移率下降，过表达NFE2L2增加了细胞迁移率（P <0.0001 ）；（9）LPS处理后，自噬标志物的表达下降，转染NFE2L2质粒后，自噬标志物的表达增加，但加入pcDNA-KEAP1后，自噬标志物的表达减少（P < 0.05）。结论 发现了NFE2L2/KEAP1在牙周炎中的调控作用，为治疗牙周炎提供科学依据。 -
关键词:
- 牙周炎 /
- KEAP1/NFE2L2 /
- 自噬 /
- 成骨细胞
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. -
Key words:
- Periodontitis /
- KEAP1/NFE2L2 /
- Autophagy /
- Osteoblasts
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图 1 各组病理变化（标尺 = 50 μm，×200）
Figure 1. Pathological changes in each group（scale bar = 50 μm，×200）
图 2 各组破骨细胞阳性率（标尺 = 50 μm，×200）
A：破骨细胞阳性率；B：破骨细胞阳性率统计；****P <
0.0001 。Figure 2. Osteoclast positivity rate in each group （scale bar = 50 μm，×200）
图 3 各组炎症因子
A：IL-1β的浓度；B：IL-10的浓度；C：Tnf-α的浓度；*P < 0.05，**P < 0.01，***P < 0.001，****P <
0.0001 。Figure 3. Concentrations of inflammatory factors in each group
图 4 免疫荧光以及qPCR检测NFE2L2的表达
A：免疫荧光检测NFE2L2的相对荧光强度（scale bar = 100 μm，×100）；B：免疫荧光检测平均荧光强度的统计量化结果；C：qPCR检测NFE2L2的mRNA表达；***P < 0.001，****P <
0.0001 。Figure 4. NFE2L2 expression detected via immunofluorescence and qPCR
图 5 各组成骨蛋白标志物的蛋白表达
A：WB条带；B：ALPL2相对蛋白表达；C：RUNX2相对蛋白表达；D：COL1相对蛋白表达；*P < 0.05，**P < 0.01，***P < 0.001，****P < 0.0001。
Figure 5. Protein expression of osteogenic protein markers
图 6 NFE2L2过表达质粒转染效率
A：WB条带；B：NFE2L2相对蛋白表达；*P < 0.05，**P < 0.01。
Figure 6. Transfection efficiency of NFE2L2 overexpression plasmid
图 7 NFE2L2过表达质粒转染后细胞活性
A：通过显微镜观察原代细胞牙周韧带细胞（hPDLCs）的形态（scale bar = 100 μm，×100）；B通过CKK-8观察细胞的增殖；****P < 0.0001。
Figure 7. Cell viability after transfection with NFE2L2 overexpression plasmid
图 8 NFE2L2过表达质粒转染后钙化结节情况（标尺 = 200 μm，×50）
Figure 8. Calcified nodule formation after NFE2L2 plasmid overexpression （scale bar = 200 μm，×50）
图 9 NFE2L2过表达质粒转染后成骨标志物的蛋白表达
A：WB条带；B：ALPL2相对蛋白表达；C：RUNX2相对蛋白表达；D：COL1相对蛋白表达；*P < 0.05，**P < 0.01，***P < 0.001。
Figure 9. Protein expression of osteogenic markers after NFE2L2 plasmid overexpression
图 10 NFE2L2过表达质粒转染后细胞迁移检测（标尺 = 100 μm，×100）
A：细胞0 h以及24 h时划痕图；B：细胞迁移率；****P <
0.0001 。Figure 10. Cell migration assay after transfection after NFE2L2 plasmid overexpression （scale bar = 100 μm，×100）
图 11 转染NFE2L2和KEAP1质粒后NFE2L2的蛋白表达
A：WB条带；B：NFE2L2相对蛋白表达；*P < 0.05，**P < 0.01。
Figure 11. Protein expression of NFE2L2 after transfection with NFE2L2 and KEAP1 plasmids
图 12 转染NFE2L2和KEAP1质粒后自噬标志物的表达
A：WB条带；B：Beclin1 相对蛋白表达；C：LC3II/LC3I；D：ATG5相对蛋白表达；*P < 0.05，**P < 0.01，***P < 0.001，****P < 0.0001。
Figure 12. Expression of autophagy markers after transfection of NFE2L2 and KEAP1 plasmids
图 13 转染NFE2L2和KEAP1质粒后成骨蛋白标志物的蛋白表达
A：WB条带；B：ALPL2相对蛋白表达；C：RUNX2相对蛋白表达；D：COL1相对蛋白表达；*P < 0.05，**P < 0.01，***P < 0.001。
Figure 13. Protein expression of osteogenic protein markers after transfection with NFE2L2 and KEAP1 plasmids
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