The Regulatory Mechanism Of Epithelial Junction Disruption By Porphyromonas Gingivalis-induced Abnormal Iron Metabolism In Oral Epithelial Cells

Objective:Periodontitis is a chronic infectious disease that occurs in the periodontal supporting tissues initiated by plaque biofilm.The oral epithelium is the first line of defense against pathogens invading periodontal tissues,and the junction of oral epithelial cells plays an important and central role.In the state of periodontal inflammation,periodontal pathogens can invade gingival tissues and deeper tissues by destroying epithelial cell junctions.As a key periodontal pathogen,Porphyromonas gingivalis（P.gingivalis）can disrupt epithelial junctions,invade the epithelial barrier and exacerbate periodontal tissue destruction through multiple pathways.Therefore,it is of great significance to deeply study the etiology of periodontitis to explore the mechanism of P.gingivalis infection destroying the oral epithelium connection.Iron（Fe）is a trace metal essential to ensure the survival of almost all organisms.Its participation in a variety of vital functions,including oxygen transport,DNA synthesis,cellular respiration and metabolic energy.As one of the important metabolic pathways of life,the homeostasis of iron metabolism is crucial for the body to maintain normal physiological functions,and abnormal iron metabolism can cause pathological changes.Iron overload can lead to the generation of reactive oxygen species,which triggers oxidative stress,lipid peroxidation,and even DNA damage.Meanwhile,iron element is an exogenous nutrient element necessary for the survival of P.gingivalis.P.gingivalis can take up and store iron.Studies have shown that P.gingivalis can affect host cell iron metabolism by synthesizing and secreting proteases to ingest iron in host cell ferritin.The previous study of our group suggested that P.gingivalis infection may lead to abnormal iron metabolism of oral epithelial cells,resulting in the destruction of epithelial junctions,leading to further aggravation of periodontal damage.However,there are few reports on the impact of abnormal iron metabolism on the oral epithelial barrier.Based on the above research background,this study combined bioinformatics methods to screen iron metabolism-related molecules,detected the expression levels of iron metabolism and epithelial junction-related proteins through animal experiments and in vitro experiments,and established an oral epithelial cell infection model to explore the effects of P.gingivalis infection on oral epithelial cells.The molecular mechanism of epithelial junction damage caused by abnormal iron metabolism,and the expression and enrichment of iron metabolism molecules were verified by transcriptome sequencing in order to further explore the molecular mechanism of periodontitis and provide new ideas for clinical treatment targets of periodontitis.Methods:This research is mainly divided into the following three parts:Part 1:（1）Screened eligible datasets of clinical periodontitis gingival tissue gene expression from the Gene Expression Omnibus（GEO）and obtained epithelial junction-related genes and ferroptosis-related genes through literature review and databases respectively.The differentially expressed genes related to epithelial junction and iron metabolism in periodontitis tissues were obtained.The correlation between epithelial junction and iron metabolism genes was analyzed by Spearman correlation analysis.（2）Established a rat model of experimental periodontitis.Hematoxylin-eosin staining and Micro-CT were used to determine the level of inflammation in rat periodontal tissues,and immunohistochemical staining was used to detect the distribution and expression of epithelial junctions proteins occludin（OCLN）,cadherin 1（E-cadherin,CDH1）,zonula occludens 1（ZO-1）and iron metabolism-related protein,ferrtin light chain（FTL）,glutathione peroxidase（Glutathione peroxidase 4,GPX4）in healthy and diseased gingiva tissues of rats.Part II:Constructed the vitro model of oral epithelial cells infected by P.gingivalis ATCC 33277 or Streptococcus gordonii（S.gordonii）strain S.gordonii Challis CH1,respectively.Iron chelator（deferoxamine,DFO）was used to intervent the iron metabolism in oral epithelial cells to clarify the effect of P.gingivalis infection on the expression of epithelial junction-related proteins.（1）CCK8 was used to detect cell proliferation ability;（2）transmission electron microscope was used to observe cell ultrastructure and mitochondrial morphological changes;（3）flow cytometry was used to detect intracellular lipid peroxidation level;（4）fluorescent probes were used to detect intracellular iron levels;（5）ELISA was used to detect intracellular glutathione content and peroxidase activity;（6）wetern blot was used to detect the protein expression level of OCLN,CDH1,FTL,GPX4 and SLC7A11 in cells.Part III:（1）Constructed a model of oral epithelial cells infected by P.gingivalis.Performed transcriptome sequencing of both experimental group and control group and screened the key regulatory molecules that caused abnormal iron metabolism in oral epithelial cells infected by P.gingivalis.The expression of key molecules was verified by qRT-PCR.（2）When the key regulatory molecule transcription factor MYB was inhibited by si RNA,the gene expression level of SLC7A11 and GPX4 and the protein expression level of CDH1,OCLN,SLC7A11 and GPX4 were detected by qRT-PCR and western blot,respectively,to explore the regulatory relationship between MYB,SLC7A11 and GPX4.（3）Constructed a rescue model by transfecting si-MYB and applying the inhibitor of glutathione peroxidase.The expression of CDH1,OCLN of model cells were detected by western blot for further clarification of the molecular regulatory mechanism of the MYB/SLC7A11/GPX4 in the destruction of epithelial junctions in oral epithelial cells infected with P.gingivalis.Results:In the first part of the study we found that:（1）Bioinformatics analysis showed that the key molecules of epithelial junction in periodontitis gingival tissues were cadherin,occludin,and tight junction protein,and the key molecules related to iron metabolism were glutathione peroxidase and ferritin with a close correlation（|r|>0.5）;（2）Compared with the control group,the expression of epithelial junction protein cadherin,occludin,and tight junction protein and iron metabolism-related protein GPX4 in rat periodontitis gingival tissues were significantly decreased,and the expression of ferritin was significantly increased（P<0.05）.In the second part of the study we found that:（1）The ability of proliferation of infected oral epithelial cells was significantly suppressed at high multiplicity of infection of P.gingivalis（P<0.05）;（2）Morphological changes of ferroptosis occurred in the mitochondria of oral epithelial cells after P.gingivalis infection;（3）Flow cytometry results showed that P.gingivalis infection increased the level of intracellular lipid peroxidation in oral epithelial cells,and DFO intervention significantly reduced the abnormal increase caused by P.gingivalis infection（P<0.05）;（4）The results of immunofluorescence showed that P.gingivalis infection could increase the content level of Fe²⁺in intracellular and mitochondrial in oral epithelial cells.DFO intervention significantly attenuated the abnormal increase of Fe²⁺content（P<0.05）;（5）ELISA results showed that P.gingivalis infection could effectively reduce the level of glutathione in oral epithelial cells.DFO intervention significantly attenuated the abnormal decrease（P<0.05）;（6）Western blot results showed that in the 24 h short-term P.gingivalis infection model,the expression of iron-promoting molecules was increased,the expression of iron-inhibiting molecules was decreased,and the expression of epithelial connexin was decreased（P<0.05）;（7）Contrasted the infection model of P.gingivalis or S.gordonii,we found that the infection pattern of P.gingivalis was specific.While the S.gordonii infection increased epithelial connexin expression and inhibited iron metabolism,the P.gingivalis infection decreased epithelial connexin expression and promoted iron metabolism（P<0.05）.The results showed that P.gingivalis infection could specifically lead to abnormal iron metabolism and weaken the epithelial junctions of oral epithelial cells.In the third part of the study we found that:（1）Transcriptome Sequencing results showed that TWIST1,SOX4,SMAD3,MYB,and P53 were highly expressed among the differentially expressed transcription factors,and MYB had the most significant difference in expression（FC=2.387,P<0.05）;（2）After silencing MYB,the m RNA and protein expression level of glutathione peroxidase and SLC7A11 were significantly increased（P<0.05）,and the protein expression level of cadherin and occludin were significantly increased（P<0.05）.The results suggested that MYB could effectively inhibit the expression of SLC7A11,glutathione peroxidase and oral epithelial junction molecules;（3）glutathione peroxidase inhibitors could significantly attenuate the increase of the expression of cadherin and occludin caused by si-MYB（P<0.05）,suggesting that glutathione peroxidase could block the inhibition of MYB on the expression of oral epithelial junction molecules to a certain extent.Therefore,P.gingivalis may promote the disruption of oral epithelial cell junctions mediated by iron metabolism disorder through the MYB/SLC7A11/GPX4 axis.In conclusion:1.P.gingivalis induced oral epithelium iron overload and weakened the epithelial junctions.2.The iron chelator DFO could attenuate the iron metabolism disorder and restore the epithelial junctions of oral epithelial cells induced by P.gingivalis to a certain extent.3.Compared with S.gordonii,the iron metabolism disorder in oral epithelial cells induced by P.gingivalis was specificity.4.P.gingivalis might promote the destruction of oral epithelial cell junctions mediated iron metabolism disorder through the MYB/SLC7A11/GPX4 axis.