国际口腔医学杂志

国际口腔医学杂志 ›› 2025, Vol. 52 ›› Issue (2): 183-194.doi: 10.7518/gjkq.2025047

• 论著 • 上一篇    下一篇

具核梭杆菌经铁死亡途径破坏体外肠道上皮屏障模型的研究

张潇月1(),陈舒泽1,周婕妤1,程磊2,赵蕾1()   

  1. 1.口腔疾病防治全国重点实验室;国家口腔医学中心 国家口腔疾病临床医学研究中心;四川大学华西口腔医院牙周病科 成都 610041
    2.口腔疾病防治全国重点实验室;国家口腔医学中心 国家口腔疾病临床医学研究中心;四川大学华西口腔医院牙体牙髓病科 成都 610041
  • 收稿日期:2024-05-24 修回日期:2024-09-21 出版日期:2025-03-01 发布日期:2025-03-01
  • 通讯作者: 赵蕾
  • 作者简介:张潇月，医师，硕士，Email：xiaoyuezhangxxx@163.com
  • 基金资助:
    国家自然科学基金(81970944);四川省自然科学基金(2023-NSFSC0553);国家自然科学基金青年项目(82301089)

Studying how Fusobacterium nucleatum affects the intestinal epithelial barrier model in vitro via the ferroptosis pathway

Xiaoyue Zhang1(),Shuze Chen1,Jieyu Zhou1,Lei Cheng2,Lei Zhao1()   

  1. 1.State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Dept. of Periodontology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
    2.State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Dept. of Cariology and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
  • Received:2024-05-24 Revised:2024-09-21 Online:2025-03-01 Published:2025-03-01
  • Contact: Lei Zhao
  • Supported by:
    National Natural Science Foundation of China(81970944);Natural Science Foundation of Sichuan Province(2023NSFSC0553);National Natural Science Foundation of China Youth Program(82301089)

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摘要：

目的 探究具核梭杆菌（F. nucleatum）感染对体外肠道上皮屏障的影响及可能的机制。 方法 人结直肠腺癌细胞Caco-2细胞接种于Transwell小室构建体外肠道上皮屏障模型。采用葡聚糖硫酸钠（DSS）和F. nucleatum建立细胞损伤和感染模型，实验分为CON组、FN组、8%DSS组和FN+8%DSS组，检测F. nucleatum对有无DSS处理的上皮屏障的影响并评估铁死亡在其中的作用；随后引入铁死亡抑制剂铁抑素1（Fer-1）和去铁胺（DFO），探究抑制铁死亡对受损上皮屏障的作用。实验中，采用细胞计数试剂盒8（CCK-8）检测细胞增殖；乳酸脱氢酶（LDH）法检测细胞损伤；跨上皮电阻值（TEER）评估上皮完整性；异硫氰酸荧光素-葡聚糖（FD4）透过率评估上皮通透性；透射电镜观察细胞间连接及线粒体；蛋白免疫印迹和免疫荧光染色检测胞质紧密粘连蛋白ZO-1和紧密连接蛋白CLDN-1的表达；铁死亡检测包括免疫荧光染色检测细胞内亚铁离子（Fe2+）含量；免疫印迹法检测谷胱甘肽过氧化物酶4（GPX4）、铁蛋白重链1（FTH1）、乙酰辅酶A合成酶长链家族4（ACSL4）的表达，以及测量丙二醛（MDA）含量和谷胱甘肽比例（GSH%）评估脂质过氧化水平。 结果 与CON组相比，FN组、8%DSS组和FN+8%DSS组细胞增殖率降低，细胞损伤增加（P<0.05）；与CON组相比，FN组TEER值和FD4透过率差异无统计学意义（P>0.05）；与8%DSS组相比，FN+8%DSS组的6、12、24 h TEER值下降（P<0.05），FD4透过率增加（P<0.000 1）；与CON组相比，FN+8%DSS组ZO-1和CLDN-1蛋白下调（P<0.05）；透射电镜观察到8%DSS组细胞间连接破坏，FN+8%DSS组除了细胞间连接破坏还观测到F. nucleatum入侵细胞内部，线粒体呈现铁死亡样改变；GPX4蛋白下调，FTH1和ACSL4蛋白上调，细胞内Fe2+积聚，MDA升高，GSH%降低。引入Fer-1和DFO后，细胞损伤降低，TEER值回升，FD4透过率降低，ZO-1和CLDN-1蛋白表达升高（P<0.05）。 结论 F. nucleatum感染可能通过铁死亡途径促进DSS诱导的体外肠道上皮屏障破坏。

关键词: 具核梭杆菌, 肠道上皮屏障, 铁死亡, Caco-2细胞, 牙周炎, 溃疡性结肠炎

Abstract:

Objective This study investigates the effect of Fusobacterium nucleatum (F. nucleatum) infection on the intestinal epithelial barrier in vitro and possible mechanisms. Methods An in vitro intestinal epithelial barrier model was established by inoculating human colorectal adenocarcinoma cells (Caco-2 cells) in Transwell chambers. Dextran sodium sulfate (DSS) and F. nucleatum were used in establishing a cell injury and infection model, and the experimental groups were divided into CON, FN, 8% DSS, and FN+8% DSS groups, where the effects of F. nucleatum on the epithelial bar-riers with and without DSS treatment were detected and the role of ferroptosis in these effects were assessed. Subsequently, the role of ferroptosis inhibition on the damaged epithelial barrier was explored after the introduction of the ferroptosis inhibitors ferrostatin-1 (Fer-1) and deferoxamine (DFO). In the experiments, cell proliferation was detected using cell coun-ting kit 8, and cell damage was detected using lactate dehydrogenase. Epithelial integrity was assessed by trans-epithelial electrical resistance value (TEER). Epithelial permeability was assessed through fluorescein isothiocyanate-dextran (FD4) transmittance, and intercellular junctions and mitochondria were observed through transmission electron microscopy. Western blotting and immunofluorescence staining were used in detecting the expression levels of zonula occludens-1 (ZO-1) and claudin-1 (CLDN-1). Ferroptosis assay included the immunofluorescence staining of intracellular ferrous iron (Fe2+), Western blotting of glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), and acetyl-coenzyme A synthetase long-chain family 4 (ACSL4) expression, and measurement of malondialdehyde (MDA) and glutathione ratio (GSH%) to assess the lipid peroxidation levels. Results Cell proliferation rate decreased, and cell damage increased in the FN, 8% DSS, and FN+8% DSS groups compared with the CON group (P<0.05). No significant differences in TEER values and FD4 permeability were found between the FN and CON groups. TEER values at 6, 12 and 24 h decreased in the FN+8% DSS group compared with the 8% DSS group (P<0.05), and FD4 permeability increased (P<0.000 1). ZO-1 and CLDN-1 proteins were down-regulated in the FN+8% DSS group compared with the CON group (P<0.05). Transmission electron microscopy results showed disruption in intercellular junctions in the 8% DSS and FN+8% DSS groups. F. nucleatum invasion was observed, and mitochondria showed ferroptosis-like alterations. GPX4 protein was down-regula-ted, FTH1 and ACSL4 proteins were up-regulated, intracellular Fe2+ accumulated, MDA level was elevated, and GSH% was reduced. The introduction of Fer-1 and DFO reduced cellular damage and FD4 permeability, resulted in the rebound of TEER va-lues, and elevated the levels of ZO-1 and CLDN-1 proteins (P<0.05). Conclusion F. nucleatum infection may promote DSS-induced gut epithelial barrier disruption in vitro through the ferroptosis pathway.

Key words: Fusobacterium nucleatum, intestinal epithelial barrier, ferroptosis, Caco-2 cell, periodontitis, ulce-rative colitis

中图分类号: 

  • R780.2

图1

Caco-2上皮单层细胞增殖和损伤分析A、B：CCK-8和LDH法分别测定不同MOI的F. nucleatum和不同质量分数DSS感染下的细胞增殖率和LDH 释放率；C、D：CCK-8 和LDH法测定不同组别的细胞存活率和LDH释放率；E：Caco-2细胞形态 光镜 × 100。**P<0.01，***P<0.001，****P<0.000 1。"

图2

Caco-2上皮单层屏障功能评估A：Caco-2 细胞培养21 d 后分化成单层结构 光镜 × 200；B：4组细胞的FD4透过率；C：4组细胞的TEER值。*P<0.05，***P<0.001，****P<0.000 1。"

图3

Caco-2上皮单层的透射电镜观察上：× 10 000；下：× 20 000；黑色箭头示细胞间连接，白色箭头示侵入细胞内的F. nucleatum菌体。"

图4

Caco-2上皮单层紧密连接蛋白的表达分析A：ZO-1和CLDN-1的蛋白免疫印迹结果；B：ZO-1和CLDN-1蛋白免疫印迹的定量结果，α-tubulin作为内参，柱状图显示ZO-1和CLDN-1条带灰度值与ɑ-tubulin条带灰度值的比值；C：ZO-1的免疫荧光染色 × 100；D：CLDN-1的免疫荧光染色 × 100；E：ZO-1免疫荧光染色半定量分析；F：CLDN-1免疫荧光染色半定量分析。*P<0.05，**P<0.01，****P<0.000 1。"

图5

Caco-2上皮单层铁死亡评估A：GPX4、FTH1和ACSL4的蛋白免疫印迹电泳图；B：蛋白免疫印迹相对定量分析，ɑ-tubulin作为内参，柱状图显示GPX4、FTH1和ACSL4与ɑ-tubulin条带灰度值的比值；C：Caco-2上皮单层细胞的Fe2+信号 免疫荧光染色 × 100； D：Caco-2上皮单层的GSH%；E：Caco-2上皮单层MDA表达水平；F：Caco-2上皮单层的透射电镜图像（上：× 10 000，下：× 20 000），黄色箭头示线粒体外膜破裂，线粒体嵴减少或消失，蓝色箭头示线粒体异常凝聚，膜密度增加。***P<0.001，****P<0.000 1。"

图6

引入铁死亡抑制剂后Caco-2上皮单层屏障功能评估A：测定 Caco-2 上皮单层的 LDH 释放率；B：F. nucleatum和8%DSS共培养24 h后FD4透过率；C、D：F. nucleatum和8%DSS共培养6、12、24、48 h后Caco-2上皮单层的TEER值，其中C为引入Fer-1，D为引入DFO；E：ZO-1免疫荧光染色 × 100；F：CLDN-1免疫荧光染色 × 100；G：ZO-1免疫荧光染色的半定量分析；H：CLDN-1免疫荧光染色的半定量分析。*P<0.05，**P<0.01，***P<0.001，****P<0.000 1。"

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