| [Purpose]To explore their differential metabolites and related metabolic pathway changes,and to explain the biological mechanism of diarrhea disease with greasy coating,metabolomics technology was used to study the metabolic differences of tongue coating and feces between cold-damp diarrhea with white greasy coating and damp-heat diarrhea with yellow greasy coating.[Methods]Part 1 Non-targeted metabolomics study of tongue coating metabolites based on UPLC-MS technology:From June 2019 to December 2019,11 patients with white greasy coating(BN group)and 12 patients with yellow greasy coating(HN group)from the Department of Spleen,Stomach,Liver and Gallbladder Diseases affiliated to Jiangxi University of Chinese Medicine were selected as the experimental group.Taking 12 healthy people with thin white coating(ZC group)in the physical examination department as the control group,UPLC-MS was used to conduct non-targeted metabolomics experiments to detect three groups of tongue coating metabolites,using the principal components Statistical methods such as Principal Component Analysis(PCA),Orthogonal Partial Least Squares Discrimination Analysis(OPLS-DA),Difference analysis,Cluster analysis and other statistical methods were used to perform quality control analysis and multivariate statistical analysis on the obtained non-targeted metabolomics data,screen metabolic markers,enrich and analyze their metabolic pathways.Part 2 Targeted metabolomics study of tongue coating and fecal metabolites based on GC-MS technology: From June 2020 to June 2021,the tongue coating(TBN group)and feces(FBN group)of patients with cold-damp diarrhea and white greasy coating from the department of Spleen,Stomach,Liver and Gallbladder Diseases,Affiliated Hospital of Jiangxi University of Chinese Medicine,and the tongue coating of patients with damp-heat diarrhea with yellow greasy coating(THN group)and feces(FHN group),13 cases each with thin white coating and tongue coating(TZC group)and feces(FZC group)of healthy individuals in the physical examination department.Targeting metabolites,detecting six groups of tongue coating and feces,using PCA analysis,Partial Least Squares Discrimination Analysis(PLS-DA),Difference analysis,Cluster analysis and other multivariate statistical analysis methods to screen metabolic markers and explore their metabolic pathways.Results:1 Non-targeted metabolomics study1.1 Quality control analysis: After the principal component analysis of the overall samples and the quality control samples,it can be proved that the quality control samples are authentic and reliable.the quality control is successful and the obtained non-targeted metabolomics data is of good quality,and the constructed data model is stable and reliable.1.2 Cluster analysis:(1)The samples in each group have a large correlation and a small degree of dispersion;(2)The distinction between the BN group and the ZC group,and between the HN group and the ZC group is good;(3)The inter-group correlation between the BN group and the HN group is higher.duration of peaks in the BN group and the HN group were significantly more than those in the ZC group.Suggesting that the metabolism of BN group and HN group during these two time periods,The species and abundance of the compounds were significantly different from those in the ZC group;(2)Within 5-6 min,there were differences in the spectral peaks between the BN group and the HN group,suggesting that there were certain differences in their metabolites.1.4 Multivariate statistical analysis:(1)The constructed models of the BN group and the ZC group,the HN group and the ZC group were well distinguished,robust and reliable,with high cumulative interpretation rate and high prediction rate.There were significant differences between the ZC groups;(2)The constructed models of the BN group and the HN group overlapped more,and the classification effect was poor,meaning which had a statistical difference.1.5 Difference analysis:(1)There were 221 types of different metabolites between the BN group and the ZC group,of which 23 types were significantly different metabolites,which were 4 types with significantly increasing expression,such as Dodecanedioic acid,3-glucoside and(-)-Fumigaclavine B and Arginyl-Phenylalanine,etc;and 19 kinds of metabolites were significantly descending expression,such as N(6)-Methyllysine,CDP-DG(a-17:0/i-24:0),Ganoderic acid H,17,18-Di HETE,Ganglioside GM2(d18:0/12:0),Azukisaponin III,Ganodericacid Ma,Ganglioside GD3(d18:0/12:0),20-Oxo-leukotriene E4,Thromboxane B2,Glutamylarginine,Sericoside,Isoleucylproline,oxirene-2,3-diol,Lubiprostone,Notoginsenoside T1,5’-Carboxy-gamma-chromanol,3,4-dihydroxybutanoic acid,Goshonoside F5,etc.Significantly different metabolites belong to the fat metabolism pathway;(2)There were 231 kinds of different metabolites between the HN group and the ZC group,of which 27 types were significantly differential1.3 Metabolic fingerprints:(1)Within 0-5min and 6-8min,the number and metabolites,which were 7 kinds of metabolites with significantly decreased expression,such as Dodecanedioic acid,3-glucoside,Hesperidin,(±)10-HDo HE,Pyroglutamyl-prolyl-arginine-4-nitroanilide,7E,9E-Octadecadienoic acid,Cis-5-dodecenoic acid,etc,and 20 kinds of metabolites were significantly descending expression,such as N(6)-Methyllysine,Ganglioside GM2(d18:0/12:0),17,18-Di HETE,CDP-DG(a-17:0/i-24:0),Ganodericacid Ma,Azukisaponin III,Ganglioside GD3(d18:0/12:0),20-Oxo-leukotriene E4,Thromboxane B2,Glutamylarginine,Sericoside,Isoleucylproline,Norerythromycin,PI(16:0/16:1(9Z)),oxirene-2,3-diol,Lubiprostone,Notoginsenoside T1,5’-Carboxy-gamma-chromanol,Arginyl-Phenylalanine,3,4-dihydroxybutanoic acid,etc.Most of the significantly different metabolites belong to the fat metabolism pathway;(3)20 metabolites with significant difference between the BN group and the ZC group were the same as those between the HN group and the ZC group;(4)There were 39 different metabolites between the BN group and the HN group,of which there were7 different metabolites with statistical significance: Hesperidin,Berberine,Nobiletin,Tangeritin,S-(Hydroxymethyl)glutathione,Apigenin,Daidzein,etc.Hesperidin,Nobiletin,Tangeritin,Apigenin,and Daidzein belong to flavonoids,with yellow color or yellow-green color.Berberine is an alkaloid with strong yellow color.Apigenin was associated with apoptosis,and the remaining six differential metabolites were all associated with inflammation.1.6 KEGG enrichment analysis of differential metabolites:(1)Compound classification analysis: The differential metabolites between the BN group and the ZC group mainly belonged to two types of lipids,phospholipids and eicosanoids,and the main differential metabolites between the HN and ZC groups were they belonged to two types of phospholipids and organic acids,while the differential metabolitesbetween BN group and HN group mainly belonged to flavonoids;(2)Pathway cluster analysis: there were significant differences in the metabolic pathways between the BN group and the ZC group,and between the HN group and the ZC group,which were mainly enriched in the first to fourth levels,and enriched many metabolic pathways.The differences in metabolic pathways between the groups were not significant,and were mainly manifested in the two metabolic pathways in the first to fourth levels;(3)Metabolic pathway analysis: The relatively more important and credible metabolic pathway changes between the BN group and the ZC group,and between the HN group and the ZC group were the down-regulated expression of the ether lipid metabolism pathway under the lipid metabolism pathway,and the ether lipid and unsaturated fatty acids were down-regulated.The unsaturated ether phospholipids formed by binding are important regulators of cell sensitivity to ferroptosis.The down-regulation of ether lipid metabolism pathway is closely related to the decrease of cell sensitivity to ferroptosis.(4)The most important metabolic pathway between the BN group and the HN group was the isoflavone biosynthesis pathway.2 Targeted metabolomics study2.1 Differential Metabolite Screening: The differential metabolites screened out by multivariate statistical analysis were C12:0(lauric acid),C13:0(tridecanoic acid),C16:1(palmitate),C17:0(heptadecanoic acid),C18 :0(stearic acid)and its isomers(C18:1n9c,C18:2n6c,C18:3n3),C20:0(arachidonic acid),C22:0(behenic acid),C24:0(four dodecanoic acid,0.54),The following are the specific abundance changes of the differential metabolites screened between each group(Note: numbers in brackets are FC values):(1)Between the TBN group and the FBN group:C16:1(1.86),C17:0(1.84),C18:0(1.50),C18:1n9c(0.77),C18:2n6c(0.62);(2)Between THN group and FHN group: C12:0(1.56),C22:0(0.53),C24:0(0.54);(3)Between TZC group and FZC group: C13:0(4.40),C16:1(3.16),C17:0(3.05),C18:1n9c(0.69),C18:2n6c(0.46);(4)TBN group and TZC group Between groups: C20: 0(1.75);(5)Between THN group and TZC group: C16: 1(1.25),C17: 0(1.26),C18: 2n6c(0.70),C18: 3n3(0.31);(6)TBN group Between group and THN group: C16:1(0.81),C17:0(0.80),C18:3n3(2.69);(7)FBN group and FZC group: C16:1(0.60),C20:0(0.60),C22: 0(0.54),C24:0(0.54);(8)Between FHN group and FZC group: C16:1(0.5),C17:0(0.54),C18:2n6c(1.30);(9)No screening between FBN and FHN groups differential metabolites;(10)The content of medium and long-chain fatty acids in feces of patients with cold-damp diarrhea and damp-heat diarrhea is higher than that of healthy people,suggesting that there may be medium and long-chain fatty acid absorption disorders in the intestinal tract of patients.2.2 Metabolic pathway changes:through the enrichment analysis of differential metabolites KEGG metabolic pathways,the statistically significant different metabolic pathways screened out are fatty acid synthesis pathway(6 bar),unsaturated fatty acid synthesis pathway(5bar),fatty acid elongation pathway(1 bar),fatty acid degradation pathway(1).bar),phototransduction pathway(1 bar).The synthesis of unsaturated fatty acids in patients with cold-damp diarrhea and damp-heat diarrhea was significantly down-regulated,which would lead to down-regulation of the expression of ether lipid metabolism pathways.The results of this study verified and further explained the results of the first part.Conclusion:1.Non-targeted metabolomics studies have found that there are significant metabolic disorders of medium and long-chain fatty acids in the tongue coating metabolites of patients with cold-damp diarrhea and damp-heat diarrhea,and the expression abundance of medium and long-chain fatty acids is mainly down-regulated.The most important metabolic pathway change is the down-regulation of the ether lipid metabolic pathway.The metabolite of this pathway is unsaturated ether phospholipid,which is generated by the combination of ether lipid and unsaturated fatty acid,and is a key factor in regulating the sensitivity of cells to ferroptosis.The metabolism of ether lipids is down-regulated,and the synthesis of unsaturated ether phospholipids decreases,which in turn leads to a decrease in the sensitivity of tongue epithelial cells to ferroptosis.It may be due to the significant lack of death,abnormal differentiation,keratinization,and abnormal proliferation of tongue papillae in the tongue epithelial cells,which eventually lead to the greasy coating.Targeted metabolomics studies have found that the most significant metabolic pathway changes in tongue coating and fecal metabolites in patients with cold-damp diarrhea and damp-heat diarrhea are the decrease in the synthesis of unsaturated fatty acids.This discovery confirmed the results of the first part of the study.2.The most important differential metabolic pathway among the six groups is the unsaturated fatty acid synthesis pathway under the fatty acid synthesis pathway,suggesting that the synthesis of unsaturated fatty acids in patients with cold-damp diarrhea and damp-heat diarrhea is significantly down-regulated,which verifies the results of the first part of the study and is also a clinical application.The treatment of diarrhea provides a good idea. |
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