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.
