| Objective: Porphyromonas gingivalis(P.gingivalis)is a major periodontal pathogen with a variety of virulence factors.At present,studies have shown that P.gingivalis can lead to the imbalance of intestinal micro-ecology,destroy the physical barrier of intestinal epithelium,and cause intestinal inflammatory reaction.Outer membrane vesicles(OMVs)are derived from the cell membrane of gram-negative bacteria and contain a variety of functional bacterial products,including adhesins,toxins and enzymes,as well as non-protein antigens.Mitochondrial dysfunction plays an important role in inflammatory bowel disease(IBD),but it is not known whether the OMVs of P.gingivalis can promote IBD by inducing mitochondrial dysfunction of intestinal epithelial cells.The purpose of this study is to explore the new mechanism of the OMVs of P.gingivalis in promoting IBD,so as to provide new targets and ideas for revealing the interaction and mechanism of periodontitis and IBD.Methods: P.gingivalis OMVs were obtained by ultrafiltration and ultracentrifugation.The human colorectal adenocarcinoma cells(Caco-2)were treated by P.gingivalis OMVs.The m RNA and protein expression levels of inflammatory factors inducible Nitric Oxide Synthase(i NOS)、cyclooxygenase 2(COX-2)and prohibitin(PHB)were detected by quantitative real-time polymerase chain reaction(q RT-PCR)and enzymelinked immunosorbent assay(ELISA)respectively.The morphological changes of mitochondria were observed,and the levels of mitochondrial membrane potential(MMP)and mitochondrial adenosine triphosphate(ATP)were measured.The protein expression level of PHB was detected and analyzed by western blot(WB).Three databases related to micro RNA(miRDB,miRWalk and Targetscan)were used to predict the miRNA that may target PHB and the miRNA with the highest score in the cross set was selected as the verification object of this study.Co-transfect the wild-type plasmid or the plasmid with PHB binding site mutation with the mimic or mimic NC of the miRNA into the cells respectively,and verify the combination of miRNA and PHB through the double luciferase reporter gene experiment.QRT-PCR was used to detect the gene expression level of this miRNA in Caco-2 cells after treatment with P.gingivalis OMVs,and to verify the transfection efficiency of the miRNA’ mimic NC.The expression level of PHB protein after using the miRNA’inhibitor was tested by WB.The mimc and mimic NC of this miRNA were used to study its role in mitochondrial dysfunction and cellular inflammatory response.At last,t-test was used to compare the mean between the two groups,and one-way ANOVA was used to compare the mean between the multiple groups.When P<0.05,the difference was considered statistically significant.Results: 1.After being treated with P.gingivalis OMVs,the expression level of inflammatory factors(i NOS,COX-2)in Caco-2 cells increased;mitochondrial structure was damaged,with swelling,division and cristae dissolution occurring;mitochondrial function was impaired,which was manifested by the decrease of MMP and ATP levels and the m RNA and protein expression levels of PHB decreased(P<0.05).2.Mi R-216a-3p,which may target PHB,were obtained by bioinformatics method.It can directly combine with PHB,and the gene expression level increased after the stimulation of P.gingivalis OMVs(P<0.05).3.The miR-216a-3p inhibitor can make the down-regulated PHB protein level caused by P.gingivalis OMVs rise(P<0.05);miR-216a-3p mimics can aggravate mitochondrial structural damage,dysfunction and cellular inflammatory response,while the results were just the opposite after the use of inhibitor(P<0.05).Conclusions: P.gingivalis OMVs may cause mitochondrial dysfunction of intestinal epithelial cells through miR-216a-3p/PHB signal pathway,and induce or aggravate inflammation of intestinal epithelial cells.Therefore,patients with periodontitis may promote the development of IBD through the pathogenic effect of P.gingivalis. |
PDF Full Text Request