Mechanism Of Naringin And Phlorizin In The Treatment Of Ulcerative Colitis Mice Based On Widely Targeted Metabolomics

Ulcerative colitis(UC)is a globally prevalent inflammatory bowel disease.Due to the advantages of traditional Chinese medicine(TCM)with fewer side effects and a low recurrence rate,researches on the mechanism of action of TCM in the treatment of UC are gradually increasing.Previous studies have shown that naringin can reduce the inflammatory response,oxidative reaction and epithelial cell apoptosis caused by colitis.Phlorizin and naringin are structurally similar,both of which are flavonoids with antioxidant properties.However,the specific mechanism of naringin in the treatment of UC is not clear,and the research on the effect of phlorizin on UC has not been reported.Metabolomics can qualitatively and quantitatively describe the response law of biological endogenous metabolites to internal and external changes,and is widely used in the study of drug action mechanisms.Widely targeted metabolomics is a detection technology that combines the "broadness" of non-targeted metabolomics with the "accuracy" of targeted metabolomics.Widely targeted metabolomics is one of the latest developments in metabolomics research because it can be used for both qualitative and quantitative metabolites.In this study,we constructed a UC mouse model induced by sodium dextran sulfate(DSS)and detected fecal samples from mice with ulcerative colitis treated with naringin and phlorizin using ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)based on extensive targeted metabolomics.R software was used for principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)to screen the differential metabolites in the fecal samples of the control group,model group and treatment group.We analyzed the key metabolic pathways involved in differential metabolites,studied the overall metabolic changes in UC mice before and after treatment,and explored the molecular mechanism of naringin in the treatment of UC and the protective effect of phlorizin on the gut.The main research contents of this paper include:(1)Fecal samples from the control group(normal mice)and the model group(DSSinduced UC mice)were analyzed using untargeted and broadly targeted metabolomics methods.A total of 145 differential metabolites were found to be annotated by KEGG,106 differential metabolites were down-regulated and 39 differential metabolites were upregulated.After KEGG metabolic pathway enrichment analysis,15 significant metabolic pathways were found,including amino acid biosynthesis,ABC transporter,protein digestion and absorption,purine metabolism,pyrimidine metabolism,and so on.(2)Through qualitative and quantitative analysis of metabolites in the model group and naringin treatment group,a total of 26 differential metabolites were annotated by KEGG,17 differential metabolites were down-regulated,and 9 differential metabolites were up-regulated.After naringin treatment,the expression levels of 6 metabolites have been or are returning to normal.These metabolites are D-glucose-6-phosphate,prostaglandin J2,serotonin,5’-monophosphate-2’-deoxyadenosine,tryptamine,and 3-methylcatechol,which are involved in tryptophan metabolism,purine metabolism,phosphoinositide metabolism and other pathways.In this paper,the receiver operating curve(ROC curve)was used to evaluate the diagnostic accuracy of biomarkers.Among the six differential metabolites screened,prostaglandin J2(The area under the ROC curve(AUC)is 0.954)and 3-methylcatechol(AUC=0.983)showed high accuracy,sensitivity and specificity in evaluating the efficacy of naringin.(3)The test results of the model group and the phlorizin treatment group were compared and analyzed.A total of 30 differential metabolites annotated by KEGG were screened,25 differential metabolites were down-regulated and 5 differential metabolites were up-regulated.After phlorizin treatment,the expression levels of five metabolites have returned or are returning to normal.These metabolites include serotonin,tryptamine,p-toluenesulfonic acid,9E,11E-octadecadienoic acid,and(2R,3R)-3-methylglutamyl-5-semialdehyde-N6-lysine.They are involved in pathways such as tryptophan metabolism,bile secretion,and linoleic acid metabolism.9E,11E-Octadecadienoic acid(AUC=1)has high accuracy,sensitivity and specificity in evaluating the efficacy of phlorizin.(4)Comparing the phlorizin treatment group and the naringin treatment group,a total of28 differential metabolites annotated by KEGG were obtained,10 differential metabolites were down-regulated,and 18 differential metabolites were up-regulated.After KEGG metabolic pathway enrichment analysis,five significant metabolic pathways were found,including arginine and proline metabolism,estrogen signaling pathway,synaptic vesicle cycle and so on.In summary,this paper systematically studied the mechanism of naringin and phloridzin on ulcerative colitis mice through metabolomics technology: It was found that the metabolic disorder in UC mice may be caused by the imbalance of amino acid metabolism,pyrimidine metabolism and purine metabolism.Naringin has a certain curative effect on ulcerative colitis,and prostaglandin J2(AUC=0.954)and 3-methylcatechol(AUC=0.983)can be used as potential biomarkers to evaluate its curative effect.Phlorizin treatment shows a certain protective effect on the intestine,and 9E,11E-octadecadienoic acid(AUC=1)can be used as a potential biomarker to evaluate its effect.According to the treatment conditions of mice and the recovery level of metabolites after treatment with the two drugs,it is speculated that the therapeutic effect of naringin is better than that of phlorizin.The results of metabolic pathway analysis showed that the difference between the two in the treatment of UC was due to their different degrees of regulation of amino acid metabolism disorders.Therefore,this study provides research ideas and experimental basis for the effect of traditional Chinese medicine treatment on ulcerative colitis and drug development and application.