The Protective Effect And Mechanism Of Unconjugated Bilirubin On The Intestinal Mucosal Barrier

Objective:To explore the protective effect of unconjugated bilirubin(UCB)on the intestinal barrier and the level of serum unconjugated bilirubin in patients with ulcerative colitis.Method:The thesis is divided into three parts.The first part is animal experiment.20 mice were randomly divided into control group,UCB group,DSS group and DSS+UCB group.The modeling method of colitis is to give mice drinking water containing 2.5%DSS for 5 days and then return to normal water for 2 days.UCB group and DSS+UCB group were given 30mg/kg UCB by gavage,and the remaining 2 groups were gavage with distilled water for 7 days.The general condition of the mice was evaluated by the weight,the disease activity index and the length of the colon.Collect mouse colon specimens for HE staining and pathological scoring to evaluate inflammation.The expression level of tight junction protein was detected by Western blot.N-benzoyl-L-arginine ethyl ester(BAEE)was used as the substrate to determine the trypsin activity in mouse feces.The second part is the organoid experiment.The mouse colonic crypts are extracted to construct the organoid,and the TNF-? concentration gradients are set to find the appropriate dose for the establishment of the inflammation model.Set the UCB concentration gradients to explore the protective effect of different concentrations of UCB on the intestinal barrier.The permeability of FITC-dextran was used to evaluate the organoid epithelial barrier function.The third part collects clinical data to analysis.A total of 157 UC-confirmed patients admitted to the Department of Gastroenterology,Peking Union Medical College Hospital from May 2015 to May 2021 were divided into remission group,mild activity group,moderate activity group,and severe activity group according to the modified Mayo score at admission.Collect the data of serum total bilirubin,indirect bilirubin,direct bilirubin,erythrocyte sedimentation rate and high-sensitivity C-reactive protein levels at admission.Analyze the serum bilirubin levels of UC patients at different disease stages,as well as the correlation between bilirubin levels,inflammation indicators and Mayo score.Results:Animal experiment:Compared with the control group,the weight of mice in the UCB group increased significantly.Compared with the DSS group,the colon length of the DSS+UCB group was increased,and the pathological score was reduced.Western blot showed that the expression levels of Claudin-1,Claudin-2,Occludin and Zo-1 in the DSS group were lower than those in the control group,and the expression of tight junction protein increased significantly after administration of UCB.The trypsin activity in the feces of the DSS group mice was higher than that in the control group,and the trypsin activity in the UCB+DSS group was lower than that in the DSS group,but there was no significant difference.Organoid experiment:Compared with the control group,the fluorescence permeability of organoids under TNF-? stimulation increases,and it depends on the treatment time and concentration.Compared with the TNF-?group,the fluorescence permeability decreased after pretreatment with UCB,and the effect of high UCB concentration(6nM)in protecting the epithelial barrier was better than the lower concentration(2nM,4nM).Clinical part:Compared with the remission period,the indirect bilirubin level of patients in the mild,moderate and severe active period was significantly reduced,and the indirect bilirubin level was negatively correlated with the erythrocyte sedimentation rate,high-sensitivity C-reactive protein and Mayo score.Conclusion:Unconjugated bilirubin can effectively alleviate colonic inflammation in DSS mice,promote tight junction protein expression,reduce trypsin activity,and play a role in protecting the intestinal barrier.TNF-? can be used to establish organoid epithelial injury models,and UCB has a protective effect on TNF-?-induced damage.Serum UCB levels in patients with active UC are lower than those in remission,and UCB levels are negatively correlated with inflammatory indicators and modified Mayo scores,which proves the protective effect of UCB in inflammatory diseases from a clinical perspective.