| Objective:The detection of plasma and urine samples in infants with HCMV hepatitis by the methods of metabolomics was to establish a metabolite characteristic spectrum library of this disease based on differential biomarkers of Damp-heat syndrome,Pi-deficiency with dampness encumbrance syndrome,Qi stagnation and blood stasis syndrome.To clarify the characteristics of metabolic network of each TCM syndrome of infantile HCMV hepatitis,and to provide some objective basis for the study of the TCM syndromes’essence of this disease.At the same time,HCMV-induced liver injury in infants was divided into three common clinical types:HCMV hepatitis,HCMV cholestatic hepatopathy and extrahepatic biliary atresia with HCMV infection.The potential biomarkers of hepatic injury caused by HCMV were explored through metabolomic detection of plasma samples,which provided scientific basis for early diagnosis and treatment of such diseases.In addition,the detection of urine samples in infants with hepatitis syndrome and extrahepatic biliary atresia of Qi stagnation and blood stasis syndrome was to seek biomarkers of Qi stagnation and blood stasis syndrome,and to seek the biological basis of”different diseases with the same syndrome".Biomarkers of extrahepatic biliary atresia were also searched to explore the feasibility of non-invasive diagnosis of this disease.Methods:In this study,plasma and urine samples were obtained from Beijing children’s hospital affiliated to capital medical university.Metabolomics analysis of plasma and urine samples were performed by UHPLC-LTQ/Orbitrap-MS method and GC-MS method.The second part of this study was the clinical study of infants with HCMV hepatitis.The degree and duration of jaundice,clinical symptoms,and syndrome differentiation of each infant with HCMV hepatitis were listed in detail.The patients were grouped according to the inclusion and exclusion criteria.The general information and laboratory examination of each group were also analyzed.In the third part of this study,a total of 115 plasma samples of infants with HCMV hepatitis were collected and assigned to damp-heat syndrome(38 cases),Pi-deficiency with dampness encumbrance syndrome(36 cases),Qi stagnation and blood stasis syndrome(41 cases).Another 39 cases of normal control were collected at the same time.Non-targeted UHPLC-LTQ/Orbitrap-MS and GC-MS metabolomics methods were used in conjunction with PCA and OPLS-DA analysis to explore the metabolomic profiles of damp-heat syndrome,Pi-deficiency with dampness encumbrance syndrome,Qi stagnation and blood stasis syndrome.The results were analyzed by non-parametric test and Fold change.Finally,differential metabolites of these syndromes were identified by searching LipidBlast,Mona and other databases.In the fourth part of this study,a total of 122 urine samples of infants with HCMV hepatitis were collected and assigned to damp-heat syndrome(44 cases),Pi-deficiency with dampness encumbrance syndrome(35 cases),Qi stagnation and blood stasis syndrome(43 cases).Another 40 cases of normal control were collected at the same time.Non-targeted UHPLC-LTQ/Orbitrap-MS and GC-MS metabolomics methods were used in conjunction with PCA and OPLS-DA analysis to explore the metabolomic profiles of damp-heat syndrome,Pi-deficiency with dampness encumbrance syndrome,Qi stagnation and blood stasis syndrome.Finally,differential metabolites of these syndromes were identified based on the results of non-parametric test and Fold change.In the fifth part of this study,a total of 127 plasma samples of subjects were collected and assigned to HCMV hepatitis(22 cases),HCMV cholestatic hepatopathy(39 cases)and extrahepatic biliary atresia with HCMV infection(40cases).Another 40 cases of normal control were collected at the same time.Non-targeted GC-MS metabolomics methods were used in conjunction with PCA and OPLS-DA analysis to explore the metabolomic profiles of these groups.Finally,differential metabolites were identified based on the results of non-parametric test and Fold change.The metabolic pathways involved in HCMV-induced liver injury were summarized,and biomarkers of extrahepatic biliary atresia with HCMV infection were searched.In the sixth part of this study,one hundred and one urine samples of the subjects were collected and assigned to extrahepatic biliary atresia group(25 cases),infantile hepatitis syndrome(38 cases)and normal control(38 cases).A non-targeted GC-MS metabolomics method was used in conjunction with PCA and OPLS-DA analysis to explore the metabolomic profiles of different groups.Finally,differential metabolites of these syndromes were identified based on the results of non-parametric test and Fold change.Biomarkers of Qi stagnation and blood stasis syndrome were identified.Meanwhile,urinary biomarkers related to biliary atresia were searched to explore the feasibility of using metabolomics technology in non-invasive diagnosis of this disease.Results:The second part of this study found that the severity of j aundice in Qi stagnation and blood stasis syndrome was severe and lasted for a long time.Cholestatic hepatopathy,biliary atresia and cirrhosis were the common types of liver damage in this TCM syndrome.The degree of jaundice in Pi-deficiency with dampness encumbrance syndrome was slight,and the degree of jaundice in the damp-heat syndrome was different.Non-jaundice HCMV hepatitis and cholestatic hepatopathy were the common types of liver damage in these two syndromes.In the laboratory examination,hepatosplenomegaly,cirrhosis,and inadequate gallbladder filling were more common in Qi stagnation and blood stasis syndrome,which indicated that the liver injury of this syndrome is more serious.In the third part of this study,we found that HCMV hepatitis damp-heat syndrome,Pi-deficiency with dampness encumbrance syndrome,Qi stagnation and blood stasis syndrome and normal control group obviously separated in PCA and OPLS-DA model,which indicated that there were significant differences in metabolites of these groups.Totally,there were 35 differential metabolites in the three TCM syndrome groups,involving the metabolic disorders of triglyceride,sphingomyelin,phosphatidylcholine and lysophosphatidylcholine.Among them,the disorder of triglyceride metabolism was the most significant.Glycocholic acid was significantly up-regulated in Qi stagnation and blood stasis syndrome group,which was a possible biomarker of this syndrome.Melatonin was up-regulated significantly in damp-heat syndrome,which was related to the inflammatory state of this syndrome.In the fourth part of this study,we found that urine samples of different TCM syndromes of HCMV hepatitis separated clearly in PCA and OPLS-DA model,which indicated that there were significant differences in urine metabolites of these syndromes.All the syndromes involved the metabolism disorders of alanine,aspartic acid and glutamic acid,D-arginine and ornithine,histidine,arginine and proline.In the damp-heat syndrome,energy-related metabolites such as D-glucose,D-maltose and citric acid were down-regulated.In Pi-deficiency with dampness encumbrance syndrome,intestinal flora-related metabolites such aminomalonic acid,butyric acid,hippuric acid were up-regulated or down-regulated.Glycine,serine and threonine metabolism,lysine degradation and biosynthesis,and histidine metabolic pathways were involved in this group.In Qi stagnation and blood stasis syndrome group,amino acid metabolic pathway and energy-related metabolic pathways such as glyoxylic acid and dicarboxylic acid,citric acid cycle were involved.In the fifth part of this study,we divided HCMV-induced liver injury infants into HCMV hepatitis group,HCMV cholestatic hepatopathy group and extrahepatic biliary atresia with HCMV infection group based on their different clinical manifestations.By comparing with the normal control group,29 differential metabolites were finally found.A series of amin o acids,fatty acids and energy metabolism disorders were involved.In addition,we also found that five different metabolites,namely carbamic acid,glutamic acid,L-aspartic acid,L-homoserine,norepinephrine,were up-regulated in extrahepatic biliary atresia with HCMV infection group.The diagnostic model constructed by ROC curve analysis of these five metabolites can well distinguish HCMV cholestatic hepatopathy group and extrahepatic biliary atresia with HCMV infection group.The sixth part of this study describes the profiles of urine metabolism in biliary atresia and infantile hepatitis syndrome Qi stagnation and blood stasis syndrome.Biliary atresia,infantile hepatitis syndrome and normal control group separated clearly in OPLS-DA model.Qi stagnation and blood stasis syndrome involved a series of amino acids and purine metabolism disorders.In addition,ROC curve analysis showed that alpha-aminoadipic acid and N-acetyl-D-mannosamine were potential biomarkers for distinguishing biliary atresia from infantile hepatitis syndrome.Conclusion:1.Using UHPLC-LTQ/Orbitrap-MS and GC-MS techniques,we can analyze the metabolic profiles of plasma and urine samples of HCMV hepatitis with damp-heat syndrome,Pi-deficiency with dampness encumbrance syndrome,Qi stagnation and blood stasis syndrome,and can distinguish and elaborate the different metabolic characteristics of each TCM syndrome.2.According to the metabolomics detection results of plasma samples based on UHPLC-LTQ/Orbitrap-MS and GC-MS methods,different metabolic characteristics were preliminarily determined in plasma samples of each TCM syndrome of HCMV hepatitis.The metabolic disorders of triglyceride,sphingomyelin,phosphatidylcholine and lysophosphatidylcholine were involved in all the three TCM syndromes,and the metabolic disorder of triglyceride was the most significant.3.According to the metabolomics detection results of urine samples based on UHPLC-LTQ/Orbitrap-MS and GC-MS methods,different metabolic characteristics weredetermined in urine samples of each TCM syndrome of HCMV hepatitis.Amino acids metabolism disorder was involved in all the three TCM syndromes.The main manifestation of damp-heat syndrome was disorder of energy metabolism.Pi-deficiency with dampness encumbrance syndrome involved the disorder of intestinal flora related metabolites.Qi stagnation and blood stasis syndrome involved the amino acids metabolism and energy-related metabolism disorder.4.According to the metabolomics detection results of plasma samples based on GC-MS method,different metabolic characteristics were preliminarily determined in plasma samples of different kinds of HCMV-induced liver injury.Amino acids,fatty acids and energy metabolism disorders were involved in HCMV-induced liver injury.The diagnostic model constructed according to the differential metabolites can distinguish HCMV cholestatic hepatopathy group and extrahepatic biliary atresia with HCMV infection group.5.According to the metabolomics detection results of urine samples based on GC-MS method,’it was found that amid acids and purine metabolism disorders were involved in both biliary atresia and infantile hepatitis syndrome Qi stagnation an d blood stasis syndrome,which confirmed that "different diseases with the same syndrome" has its scientific connotation.The diagnostic model constructed according to the differential metabolites can distinguish biliary atresia and infantile hepatitis syndrome. |
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