Study On The Mechanism Of TRPA1 Maintaining Intestinal Epithelial Barrier Function Against Citrobacter Murine Infection

As the main organ for food digestion and absorption,the intestinal tract is inevitably exposed to a large number of pathogenic bacteria accompanied by food.And millions of people died of intestinal infections caused by foodborne pathogen each year.In recent years,the pathophysiological role of transient receptor potential channel TRPA1 in the gastrointestinal tract has attracted increasing attention,and has been considered as a potential target for the treatment of inflammatory pain and mechanical sensitivity associated with gastrointestinal diseases.However,the role of TRPA1 in host-pathogen interactions remains unknown.Therefore,the role and mechanism of TRPA1 in the process of intestinal pathogenic bacteria infection in mice were studied in this paper.In this study,we use the intestinal infection model of mice established by Citrobacter rodentium infection and combined with body weight change,fecal phenotype and colonization and proliferation of Citrobacter rodentium in the intestinal tract as the indicator to compare the susceptibility of WT mice and Trpa1-/-mice against bacterial infection.Then we compared the differences of inflammatory response and intestinal epithelial barrier function after the infection of Citrobacter rodentium by histological staining(H&E),immunofluorescence,western blotting and transmission electron microscope(TEM).At the cellular level,Caco-2 cell monolayer cultured for 21 days was used to simulate the body intestinal epithelial layer,and the effect of Trpa1 knockdown on the permeability of cell monolayer after Citrobacter rodentium infection was compared.Finally,the effects of bacterial lipopolysaccharide(LPS)and TRPA1 inhibitors on cytoskeleton and tight junctions were observed by immunofluorescence and Western blotting.The main results are as follows: 1,Trpa1 gene knockout did not affect daily food intake and fecal water content in mice compared to WT mice before Citrobacter rodentium infection.After Citrobacter rodentium infection,Trpa1-/-mice showed significantly slower weight gain,higher number of colonization and proliferation of intestinal bacteria and obvious diarrhea symptoms compared to WT mice,suggesting that Trpa1 gene knockout increased host susceptibility to Citrobacter rodentium infection.2,H&E histological staining showed that Trpa1 gene knockout did not affect the normal physiological structure of the colon tissue of mice.However,compared with WT mice,colon tissue of Trpa1-/-mice was significantly damaged and have more inflammatory cells infiltrated in the mucosa after Citrobacter rodentium infection.3,After Citrobacter rodentium infection,FITC-Dextran(FD4)was given intragastric administration.Fluorescopic observation showed that FITC-Dextran distributed more extensively in the intestinal mucosa of Trpa1-/-mice than WT mice.The content of FITC-Dextran in serum of Trpa1-/-mice was higher than that of WT mice through the Auto ELISA detecter detection.In addition,Transmission electron microscopy and Western blotting results of primary intestinal epithelial cells of mice showed that the deficiency of Trpa1 resulted in the destruction of tight junctions between epithelial cells after infection.4,It is confirmed the functional expression of TRPA1 in mouse intestinal epithelial cells and intestinal epithelioid cell line Caco-2through Immunofluorescence,RT-PCR and Calcium imaging experiments.And further experiments showed that knockdown of Trpa1 significantly increased the permeability of Caco-2 cell monolayer after Citrobacter rodentium infection.5,The Caco-2 cells were stimulated by LPS,and the distribution of tight junction proteins ZO-1 and Occludin on the cell membrane was changed by immunofluorescence observation,and the cytoskeleton was disordered.In the case of co-treatment with LPS and TRPA1 inhibitors,inhibition of TRPA1 significantly aggravated cytoskeletal disorders and the distribution of ZO-1 and Occludin.The results of western blotting further confirmed that TRPA1 inhibitors accelerated the decrease of ZO-1 and Occludin expression induced by LPS.In summary,the study infected WT and Trpa1-/-mice with Citrobacter rodentium and found that Trpa1 deficiency increased the sensitivity of the host to C.rodentium infection,leading to more severe disruption of intestinal epithelial barrier function and the tight junction between epithelial cells.This study also confirmed the functional expression of TRPA1 in mouse intestinal epithelial cells,and the TRPA1 will stabilize tight junction to maintain the permeability of intestinal epithelial cells.In this study,we demonstrated for the first time that ion channel TRPA1 protects intestinal epithelial barrier function during the host comes to infection,which is related to ability of TRPA1 to stabilize cytoskeleton and tight junction in the intestinal epithelial cells.