口腔医学研究

口腔医学研究 ›› 2025, Vol. 41 ›› Issue (2): 128-133.DOI: 10.13701/j.cnki.kqyxyj.2025.02.008

• 口腔生物学研究 • 上一篇    下一篇

探究NLRP3炎症小体对人原代牙龈成纤维细胞炎症刺激的影响

张黄, 苗瑞婧, 范旭升, 黄婕, 王永武*   

  1. 西湖大学医学院附属杭州市第一人民医院口腔科 浙江 杭州 310006
  • 收稿日期:2024-08-06 出版日期:2025-02-28 发布日期:2025-02-26
  • 通讯作者: *王永武,E-mail: wangyong208@163.com
  • 作者简介:张黄(1980~ ),男,浙江海宁人,副主任医师,学士,研究方向:口腔种植。
  • 基金资助:
    杭州市农业与社会发展科研项目(编号:2022509508)

Effect of NLRP3 Inflammatome on Inflammatory Stimulation of Primary Human Gingival Fibroblasts

ZHANG Huang, MIAO Ruijing, FAN Xusheng, HUANG Jie, WANG Yongwu*   

  1. Department of Stomatology, Hangzhou First People's Hospital Affiliated to Westlake University, Hangzhou 310006, China
  • Received:2024-08-06 Online:2025-02-28 Published:2025-02-26

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摘要： 目的: 本研究在于探究核苷酸结合寡聚化结构域样受体蛋白3(nod-like receptor protein 3, NLRP3)炎症小体在人原代牙龈成纤维细胞(human gingival fibroblasts, hGFs)炎症刺激中的潜在作用机制。方法: 使用沉默RNA(silencing RNA, siRNA)构建NLRP3沉默或阴性对照并转染至hGFs,与牙龈卟啉单胞菌(P.gingivalis, Pg)共培养诱导炎症,并给予1 μmol/L NLRP3炎症小体激动剂BMS-986299干预。使用流式细胞术检测各组细胞凋亡;使用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)检测炎症因子白细胞介素-1β(interleukin-1β, IL-1β)、白细胞介素-6(interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的含量;使用Western blot检测了NLRP3、半胱氨酸天冬氨酸蛋白酶-1前体(cysteinyl aspartate specific proteinase-1 precursors,pro-Caspase-1)、半胱氨酸天冬氨酸蛋白酶-1(cysteinyl aspartate specific proteinase-1,Caspase-1)与凋亡相关斑点样蛋白(apoptosis associated speck-like protein containing a caspase activating recruitment domain, ASC)等NLRP3炎症小体相关蛋白,与白细胞介素-1β前体(interleukin-1β precursors, pro-IL-1β)、磷酸化的核因子κB(nuclear factor kappa-B phosphorylation,p-NF-κB)/核因子κB(nuclear factor kappa-B, NF-κB)与消皮素D(gasdermin D, GSDMD)等细胞焦亡相关蛋白;免疫荧光检测NLRP3炎症小体的细胞定位。结果: 流式细胞术结果显示,NLRP3沉默能有效减少细胞凋亡;ELISA检测结果表明NLRP3沉默能降低Pg诱导的炎症因子水平;Western blot和免疫荧光结果表明NLRP3沉默可以抑制NLRP3炎症小体激活,并抑制pro-IL-1β的表达、NF-κB的磷酸化与GSDMD介导的细胞焦亡。而NLRP3沉默的效果均在NLRP3炎症小体激动剂BMS-98629使用后部分逆转。结论: NLRP3可以激活NLRP3炎症小体和炎症相关通路,同时促进hGFs细胞焦亡。

关键词: 牙龈成纤维细胞, NLRP3炎症小体, 牙龈卟啉单胞菌, 种植体周围炎

Abstract: Objective: To investigate the role and mechanism of nod-like receptor protein 3 (NLRP3) inflammasome in primary human gingival fibroblasts (hGFs). Methods: NLRP3 silencing RNA (siRNA) transfection was performed to silence NLRP3 in hGFs to induce inflammation by co-culturing with P.gingivalis (Pg) and intervened with 1 μmol/L BMS-986299, an NLRP3 agonist. Flow cytometry was used to detect apoptosis in each group. Enzyme linked immunosorbent assay (ELISA) was used to measure interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Western blot was adopted to evaluate NLRP3, cysteinyl aspartate specific proteinase-1 precursors (pro-Caspase-1), cysteinyl aspartate specific proteinase-1 (Caspase-1), apoptosis associated speck-like protein containing a caspase activating recruitment domain (ASC), Interleukin-1β precursors (pro-IL-1β), nuclear factor kappa-B phosphorylation (p-NF-κB)/nuclear factor kappa-B (NF-κB), and Gasdermins D (GSDMD). And immunofluorescence was performed to detect the NLRP3 localization. Results: Flow cytometry confirmed that NLRP3 silencing suppressed cell apoptosis. ELISA analysis demonstrated that NLRP3 silencing successfully reduced the expression of Pg-induced inflammation factors. Western blot and immunofluorescence revealed that the blocking of NLRP3 significantly reduced NLRP3 inflammatory body activation, inhibited pro-IL-1β expression, NF-κB phosphorylation, and the activity of GSDMD-mediated pyroptosis. However, this beneficial effect was partially restored with the administration of BMS-98629. Conclusion: NLRP3 activates NLRP3 inflammatory bodies and inflammation-related pathways and promotes pyrolysis in hGFs.

Key words: gingival fibroblasts, NLRP3 inflammasome, P.gingivalis, peri-implantitis