UPLC-QTOF-MS Based Plasma Metabolomic Study Of Inflammatory Bowel Disease

Inflammatory bowel disease(IBD),is a chronic intestinal disorder characterized by relapsing and remitting inflammatory course.It is well known that multiple factors such as genetic predisposition,gut microbes,antibiotic overload,and a dysregulated immune system attribute to IBD development.However,the exact etiology and molecular mechanisms underlying the chronic inflammation of the digestive tract are still undefined.Diagnosis of IBD is challenging due to non-specific initial symptoms and untypical disease manifestations similar to other diseases.So,the current diagnosis of IBD involves a multidisciplinary combination of clinical,endoscopic,and biochemical examinations.The complicated process not only increases the cost of diagnosis,but the invasive methodology also escalates technical hitches in addition to the patients’distress.Thus,identification of plasma biomarkers can assist the current diagnostic approaches to improve the accuracy and reduce the risks,discomfort,and financial burden caused by invasive examination.In this study,a DSS-induced mouse model of ulcerative enteritis was established,and a metabolomics study was performed on the plasma of the mouse model based on UPLC-QTOF-MS technology to screen the differential metabolism between the ulcerative colitis model group and the healthy control group.And we also analyzed the changes of related metabolic pathways.In addition,a UPLC-QTOF-MS~E based technique was also developed for the untargeted examination of the total lipids present in the patients’plasma with the aim to identify potential diagnostic biomarkers of IBD.Differential metabolites between the IBD patients and the healthy subjects were identified by univariate and multivariate analysis.And we also analyzed the possible changes of metabolic pathways in patients with IBD.And we compared the results of plasma metabolomics in the mouse model with the results of IBD patients,it was found that the differential metabolites of IBD patients and the differential metabolites of the mouse model were disturbed in the same metabolic pathways.Then,we analyzed the plasma metabonomics of the independent verification sample set.The results verified the good diagnostic ability of the screened differential metabolites and suggested that these metabolites can be used as potential IBD biomarkers.The main research contents and results are as follows:1.The mouse model of ulcerative colitis induced by DSS was established,and control group,model group and positive drug administration group were designed.The success of the animal model was verified from three aspects:clinical manifestation,evaluation of disease activity index and colon pathology.The results of our study showed that the mice in the control group ate normally,the fur was shiny,the growth and development was good,and the feces were normal.After 24 hours of modeling,the model group appeared mental malaise,dull fur,sparse stool and bloody stool.and the degree of bloody stools in the later period was very serious.The drug administration group appeared mental malaise,dull fur,sparse stool and bloody stool after 24 hours of modeling.And then,when treating with the drug,the general condition of the mice improved gradually,and their body weight increased,but bloody stools still appeared.Through the evaluation of colon histopathology,it was found that the colon mucosa of the model group and the administration group were damaged in different degrees,accompanied by different degrees of inflammatory cell infiltration.Compared with the model group,the colon mucosa in the treatment group was significantly improved.The results showed that inflammatory bowel disease occurred in both the model group and the drug administration group,and the positive drug mesalazine relieved ulcerative colitis.The above results verified the success of the mouse model of ulcerative colitis.2.Based on UPLC-QTOF-MS technique,the method of plasma metabonomics of ulcerative colitis model induced by DSS in mice was established.Through the metabolomic study of plasma of the DSS-induced mouse ulcerative colitis model,the different metabolites between ulcerative colitis model group and healthy control group were screened and identified,and the metabolic pathway analysis was used to identify the difference between ulcerative colitis model group and healthy control group.It was found that the selected differential metabolites changed significantly in arachidonic acid metabolic pathway,sphingolipid metabolic pathway,fatty acid metabolic pathway and glycerol phospholipid metabolic pathway.3.The method for the study of human plasma metabonomics of inflammatory bowel disease based on UPLC-QTOF-MS technique was established.Through the metabolomic study of plasma of the IBD patients,the different metabolites between the disease group and the healthy control group were screened and identified.Then we evaluated the diagnostic ability of differential metabolites by ROC curve analysis and found that 44 fat-soluble metabolites and 37 water-soluble metabolites showed high diagnostic ability(AUC≥0.80).4.We further verified the diagnostic ability of 44 fat-soluble metabolites and 37water-soluble metabolites screened in a verification sample set including 30 IBD patients and30 healthy controls.We screened a total of 13 potential plasma biomarkers of IBD,including Palmitic acid,7alpha,25-dihydroxycholesterol,20-hydroxyeicosatetraenoic(HETE),5,6-epoxy-eicosatrienoic acid(Ep ETr E),docosahexaenoic acid(DHA),9-heptadecylenic acid,Lactucaxanthin,alpha-Carotene,Traumatic acid,Neoquassin,Adrenic acid,PE(18:4/15:0)and DG(18:0e/2:0/0:0).By analyzing the metabolic pathways of the 13 potential biomarkers,we found that these differential metabolites were disturbed in the biosynthetic pathway of primary bile acid,arachidonic acid metabolic pathway,sphingolipid metabolic pathway,fatty acid extension metabolic pathway and glycerol phospholipid metabolic pathway.And we further explained their role in the development process of IBD.And compared with the results of the mouse model of ulcerative colitis plasma metabolites,the results showed that arachidonic acid metabolism,sphingolipid metabolism,fatty acid metabolism and glycerol phospholipid metabolism were closely related to IBD.Our results not only provide a solid and reliable basis for monitoring the changes of metabolites in IBD,but also provide possible drug targets for the treatment of inflammatory bowel disease in the future.