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  

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

作　　者：张潇月 陈舒泽 周婕妤 程磊[2] 赵蕾[1] Zhang Xiaoyue;Chen Shuze;Zhou Jieyu;Cheng Lei;Zhao Lei(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;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)

机构地区：[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

出　　处：《国际口腔医学杂志》2025年第2期183-194,共12页International Journal of Stomatology

基　　金：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)。

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<0.0001);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。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

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 

分 类 号：R780.2[医药卫生—口腔医学]