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  

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

作　　者：张黄 苗瑞婧 范旭升 黄婕 王永武 ZHANG Huang;MIAO Ruijing;FAN Xusheng;HUANG Jie;WANG Yongwu(Department of Stomatology,Hangzhou First People's Hospital Affiliated to Westlake University,Hangzhou 310006,China)

机构地区：[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

出　　处：《口腔医学研究》2025年第2期128-133,共6页Journal of Oral Science Research

基　　金：杭州市农业与社会发展科研项目(编号:2022509508)。

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1μmol/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-1βprecursors,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。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 1μmol/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-1βprecursors(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-1βexpression,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.

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 

分 类 号：R78[医药卫生—口腔医学]