Porphyromonas Gingivalis Outer Membrane Vesicles Aggravate The Inflammatory Response Of Intestinal Epithelial Cells By Inducing Ferroptosis

Objective:Porphyromonas gingivalis（P.gingivalis）,with a variety of virulence factors such as outer membrane vesicles（OMVs）,is the main pathogen of chronic periodontitis.P.gingivalis OMVs can prevent degradation and achieve long-distance transmission,which is more virulent than bacteria,thanks to the preservation of the vesicle membrane structure.An iron-dependent form of programmed cell death is ferroptosis that is a key factor in the emergence and progression of inflammatory disorders.Current researches have demonstrated the tight relationship between intestinal illnesses and P.gingivalis.But the reports about its OMVs inducing ferroptosis and aggravating intestinal inflammation are poor.In this study,we investigated the effect of P.gingivalis OMVs on the inflammatory response of intestinal epithelial cells,and explored the pathogenic role and mechanism of ferroptosis in intestinal epithelial inflammation,which provided new evidence for the connection between periodontitis and intestinal inflammatory diseases,and was a therapeutic target for preventing the incidence of intestinal inflammatory diseases.It offers a fresh concept for the avoidance and treatment of intestinal illnesses.Materials and Methods:1.The OMVs of P.gingivalis W83 were extracted by ultracentrifugation combined with ultrafiltration,and their concentrations were measured.Then it was recognized utilizing Nanoparticle Tracking Analysis（NTA）and Transmission Electron Microscope（TEM）.2.The mitochondrial morphology of Caco-2 was observed by TEM after being stimulated with P.gingivalis OMVs（10μg/m L）for 12 h.3.Real-time qPCR was used to measure the amounts of interleukin（IL）-6,tumor necrosis factor（TNF）-α,IL-18,and IL-1βin Caco-2 cells activated by P.gingivalis OMVs at 0,6,and 12 h.4.Once Caco-2 cells were stimulated with P.gingivalis OMVs,the presence of lactate dehydrogenase（LDH）and Fe²⁺were individually determined using the microplate technique and the ferrimazine microplate method.Thiobarbituric acid technique and microplate method,respectively,were used to measure the amounts of malondialdehyde（MDA）and glutathione（GSH）.5.After stimulation of Caco-2 cells by P.gingivalis OMVs,mRNA or protein expressions of FTH,LOX-1 and FSP1 were measured by Real-time qPCR,Western blot or immunofluorescence.6.With the treatment by the ferroptosis inhibitor Liproxstatin-1（1μmol/L）,the mitochondrial morphological changes of Caco-2 cells stimulated by P.gingivalis OMVs were observed.The mRNA expression levels of IL-6,IL-18,IL-1β,TNF-α,FTH,LOX-1and FSP1,and the contents of Fe²⁺,MDA and GSH were detected.7.SPSS Statistics 25 was used to statistically assess the experimental results,and P<0.05was used to determine statistical significance.Results:1.The OMVs were successfully extracted from P.gingivalis W83 culture medium by ultracentrifugation combined with ultrafiltration.2.The TEM results showed that the mitochondria of Caco-2 cells induced by P.gingivalis OMVs became smaller,the membrane density increased,and the number of cricriae decreased.3.The mRNA levels of inflammatory factors IL-6,TNF-α,IL-18 and IL-1βwere increased after Caco-2 cells were stimulated by 10μg/m L P.gingivalis OMVs for 6 h and 12 h（P<0.05）.4.The results of the cytotoxicity investigation revealed a considerably higher level of LDH.after P.gingivalis OMVs stimulation,and the number of cell damage and death increased（P<0.05）.5.The results of qRT-PCR and microplate assay showed that P.gingivalis OMVs enhanced FTH mRNA expression,Fe²⁺and MDA concentrations,while lowering GSH concentrations.in Caco-2 cells（P<0.05）.6.P.gingivalis OMVs lowered the protein expression of LOX-1 by Western blot and immunofluorescence,while increasing the mRNA expression of LOX-1 and decreasing the mRNA and protein expression of FSP1（P<0.05）.7.Treating with the ferroptosis inhibitor Liproxstatin-1,the mitochondrial morphology of Caco-2 cells was improved,the mRNA expression levels of IL-6,IL-18,IL-1β,TNF-αand FTH were decreased,LDH,Fe²⁺,and MDA concentrations were reduced,whereas GSH concentration was elevated.The mRNA expression level of FSP1 rose whereas LOX-1dropped（P<0.05）.Conclusion:P.gingivalis OMVs aggravate intestinal epithelial inflammation by inducing ferroptosis.