Effect Of Mitochondrial DNA On Permeability Of Caco-2 Cells And Its Mechanism

Background:Inflammmtory bowel disease(IBD)is a chronic intestinal nonspecific inflammatory disease of unknown etiology,and its pathogenesis is not very clear.Studies have shown that the increase of intestinal mucosal permeability caused by intestinal mucosal inflammation is involved in the occurrence and development of IBD.mtDNA is an important pro-inflammatory DAMPs,that can combine with TLR9 to participate in the occurrence of a variety of inflammatory diseases.It was found that mtDNA could significant increase the permeability of endothelial cells and destroy endothelial barrier through TLR9.Our study found that the level of mtDNA in plasma of patients with IBD was significantly increased,but it is not clear whether mtDNA contributes to intestinal inflammation by regulating TLR9 signaling pathway,which further affects permeability of intestinal epithelial cells and then participates in the occurrence of IBD.Therefore,on the basis of establishing the monolayer model of Caco-2 cells,we explore whether mtDNA affects the intestinal mucosal permeability by regulating the expression of TLR9 protein.Objective:To investigate the effect of mtDNA on monolayer permeability of Caco-2 cells and its mechanism.Methods:The Caco-2 cell model was prepared by using Transwell culture plate.The mtDNA was extracted from the liver of C57BL/6J mice.Then,added to the top of the Transwell culture plate were different concentrations of mtDNA(0μg/ml,1μg/ml,5μg/ml,10μg/ml)and FITC-dextran which were used as tool drugs to detect the changes of TEER and FITC-dextran content in the bottom chamber of the Transwell culture plate at different time,determining the best mtDNA concentration.Then,the experiment was divided into four groups: FITC-dextran,mtDNA+FITC-dextran,TLR9antagonists(ODN TTAGGG)+FITC-dextra,and mtDNA+ ODN TTAGGG+FITC-dextran to the top of the Transwell culture plate,to detect the changes of TEER and FITC-dextran content in the bottom chamber of the Transwell culture plate at different time.Meanwhile,Caco-2 cells were pretreated with mtDNA,ODN TTAGGG,mtDNA+ODN TTAGGG,respectively.The expression of TLR9 m RNA was detected by qRT-PCR,and the levels of TNF-ɑ,IL-6,IL-1β and IL-18 in the supernatant were detected by ELISA.Results:In comparision with the control group,with the prolongation of the action time and the increase of the concentration of mtDNA,the TEER in the monolayer of Caco-2 cells decreased and the content of FITC-dextran in the bottom chamber increased,and the TEER decreased significantly when the concentration of mtDNA was 10 μg/ml.Compared with 10 μg/ml mtDNA group,the addition of ODNTTAGGG significantly reduced the falling speed of TEER,and reduced the content of FITC-dextran in the bottom chamber,meanwile,the expression of TLR9 m RNA and the levels of inflammatory factors also declined.Conclusion:The mtDNA significantly increases the permeability of Caco-2monolayer,which may be related to the activation of TLR9 signal pathway.