| Objectives:This study attempted to clarify the expression site and expression pattern of IL-10RA in colonic epithelium of normal young mice and young mice with DSS induced inflammatory bowel disease,to confirm the protective effect of IL-10RA on the model of young mice with inflammatory bowel disease,and to explore whether the mechanism of action is related to the function of intestinal mucosal barrier through in vivo and in vitro experiments.Methods:This study consist of in vitro and in vivo experiment.Animal experiments:3-week-old C57/BL6J mice were randomly divided into control group and model group;3-week-old IL-10RA knockout mice with C57/BL6J genetic background(Il10ra-/-)were randomly divided into knockout group and knockout model group,there are ten mice in each group.Infant mice models of inflammatory bowel disease were given 4%DSS to establish:By observing the clinical manifestations of each group,the disease activity index score was performed,and by observing the pathological changes in the colon through HE staining to confirm the establishment of the infant model of inflammatory bowel disease;the colonic tissues were stained with IL-10RA immunofluorescence and immunohistochemical to determine the expression site and rule of IL-10RA in colonic epithelium,and real-time RT-PCR and Western Blot were used to detect the expression of IL-10RA in colon;the change of intestinal permeability was detected by FD4 test,;Real-time RT-PCR and Western Blot were used to detect the expression of tight junction protein Occludin and Claudin1 m RNA and proteins,so as to clarify changes in intestinal barrier.Cellular experiments,Caco-2 cell lines were cultured.Firstly,lentivirus infected plasmid was used to establish a Caco-2 cell line with IL-10RA knockdown,and the knockdown efficiency was determined by Real-time RT-PCR and Western Blot;the proliferation level of knockdown cells was detected by CCK8 assay;by measuring monolayer barrier resistance and permeability of cells and detecting m RNA and protein expression of tight junction associated proteins,in vitro intestinal epithelial barrier function was evaluated after IL-10RA knockdown.Results:The body weight of IL-10RA knockout mice was slightly lower than that of wild-type mice of the same age,but there was no statistical significance,and no changes about idiopathic enteritis;however,on the third day after 4%DSS induction,the weight of young mice in the model group began to decreased and the disease activity index increased significantly compared with the control group;the symptoms of young mice in the knockout model group appeared earlier than that in the model group,and the weight loss was more serious,the difference was statistically significant;HE results showed that the intestinal epithelium and crypt structure of the model group were damaged or even disappeared,the mucosa was thickened,a large number of inflammatory cells infiltrated into the submucosa,the intestinal wall was thickened,the congestion and edema were obvious,the intestinal structural integrity was destroyed,and the infant model of inflammatory bowel disease was successfully constructed,explained that the gross and pathological changes of the knocked out model group were more serious than that of the model group;immunofluorescence and immunohistochemistry identified the expression of IL-10RA in mouse colon epithelial cells,and found that the expression was significantly increased in the model group;real-time RT-PCR and Western Blot results of colon tissue also showed the same pattern:IL-10RA expression increased in the model group of young mice.Further detection of intestinal barrier function:The results of FD4 experiment showed that the penetration of FD4 from high to low was in the knockout model group,model group,knockout group and control group;real-time RT-PCR and Western Blot detected tight junction related proteins,and it was found that m RNA and protein expressions of Claudin1 and Occludin in colon tissue of young mice model group,especially in knockout model group,were significantly decreased compared with those in control group,and intestinal barrier was destroyed.In vitro experiments,Caco-2 cell line with IL-10RA knockdown was successfully constructed by using lentivirus infected plasmid,CCK8 results showed that IL-10RA knockdown resulted in slower cell proliferation,lower TEER value and higher FD4 transmission volume than control group;real-time RT-PCR and Western Blot results showed that the expression of tight junction proteins Claudin1 and Occludin in Caco-2 cells decreased after IL-10RA knockdown,there was a statistical difference compared with the control group.Conclusions:1.Demonstrated the localized expression of IL-10RA in colonic epithelial cells of young mice,and the protective effect on colonic epithelial barrier in a young model of DSS induced inflammatory bowel disease.2.DSS induced IL-10RA knockout mice exhibited a more severe inflammatory bowel disease phenotype,confirming that IL-10RA gene deficiency increased intestinal susceptibility to DSS damage.3.In vitro experiments with IL-10RA knockdown in intestinal epithelial cell lines showed significant impairment of intestinal epithelial cell barrier function,confirming the importance of IL-10RA in maintaining intestinal epithelial barrier function. |
