| ObjectiveTo establish the potential metabolic biomarker characteristic spectrum library of cold-damp block syndrome and poison-stagnant jaundice syndrome based on different metabolites of this disease through the detection of children's blood and urine metabolites in infantile cholestatic hepatopathy(ICH)with severe syndromes,and to provide certain objective basis for the study of the essence of ICH with severe syndromes.At the same time,animal experiments were carried out to explore the potential metabolic biomarkers of young rats with cholestasis,and we speculate the role of Lidan Heji(LDHJ)in improving cholestasis by observing the effect of LDHJ on related potential metabolic biomarkers to providing ideas for further research of LDHJ.Methods1.Theoretical research: By referring to ancient Chinese medicine books and searching the clinical research and experimental research literatures on cholestatic hepatopathy disease in "CNKI","Wanfang" and "Pub Med" in the past 10 years,this paper systematically reviewed and summarized the understanding of modern medicine and traditional Chinese medicine on the disease name,etiology,pathogenesis and treatment methods of ICH,elaborated the advantages of traditional Chinese medicine in the treatment of ICH,and put forward the concept of "ICH with severe syndrome".In addition,This paper also reviewed the application of metabolomics in objectification of diseases and syndromes,and concluded that metabolomics technology can provide objectification indicators for Traditional Chinese Medicine(TCM)dialectics,thus providing theoretical basis for the potential metabolic biomarkers of ICH with severe syndromes and the research thoughts for the possible mechanism of LDHJ to improve ICH.2.Clinical experimental studies: Blood and urine samples were collected from Wuhan Children's Hospital,Tongji Medical College,Huazhong University of Science and Technology.Metabolomics study of blood and urine samples were performed by liquid chromatography-tandem mass spectrometry(LC-MS/MS)and gas chromatography-mass spectrometry(GC-MS).In this study,the general situation,main symptoms,secondary symptoms,physical signs,laboratory indicators and syndrome differentiation of each child in ICH with severe syndromes are listed in detail.According to the inclusion and exclusion criteria,62 patients in the cold-damp block group,including 62 blood samples and 57 urine samples,48 cases in the poison-stagnant jaundice group,including 48 blood samples and 45 urine samples.In addition,there were 50 healthy infants in the normal control group at the same time,including 50 blood samples and 48 urine samples.Collecting,counting and analyzing respectively the general data,laboratory examination,metabolites result of each group.Above all,the results of blood metabolites and urine organic acid metabolites in normal group,cold-damps block group and poison-stagnant jaundice group were statistically analyzed to screen out the potential blood and urine metabolic biomarkers of cold-damp block syndrome and poison-stagnant jaundice syndrome.3.Animal experimental research: Blood samples were obtained from young SD rats and metabolites were detected by LC-MS/MS.The established cholestasis young rat model was divided into normal control group and LDHJ group of rats.Blood and liver tissue samples of rats in the three groups were collected to detect and analyze blood biochemical indexes and observe the histopathological changes of liver.In addition,LC-MS/MS technology was used to detect the metabolites in blood.According to the ratio of the metabolites to the corresponding internal standard abundances,the Chemo View 2.0.2 software was used to automatically calculate the concentrations of amino acids and acyl carnitine in the measured samples.The data were statistically analyzed by SPSS 25.0 statistical software to screen out potential biomarkers of blood metabolism in young rats with cholestasis.Compared with the potential metabolic blood metabolites of clinical ICH with severe syndrome,metabolic biomarkers that can better reflect the characteristics of cholestatic liver disease were further screened out,and the effect of LDHJ group on these metabolic biomarkers was observed,so as to speculate the mechanism of LDHJ on improving cholestasis.Results1.Objectivity indicator results of different syndromes of ICH with severe syndrome in clinical trialsIn the second part of this study,We found that in the two syndromes of ICH with severe syndrome,there was no significant difference in gender,birth,way of birth,feeding,age,weight and other general information(P >0.05).The course of disease in the group of ICH with poison-stagnant jaundice syndrome was longer than that in the group of ICH with cold-damp block syndrome(P<0.05).On laboratory metrics,the levels of total bilirubin(TBIL),direct bilirubin(DBIL),alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bile acid(TBA),?-glutamyl transpeptidase(GGT)and alkaline phosphatase(ALP)in the group of ICH with poison-stagnant jaundice syndrome were higher than those in the group of ICH with cold-damp block syndrome(P<0.05).In addition,there were statistically significant differences in blood metabolites and urinary organic acid metabolites in children with cold-damp block syndrome group,children with poison-stagnant jaundice syndrome group and normal control group(P<0.05).In the blood samples,compared with the normal control group,there were 27 kinds of blood metabolites(7kinds of amino acids and 20 kinds of carnitine)with statistical difference between severe ICH syndrome(cold-damp block syndrome and poison-stagnant jaundice syndrome)group and normal group,which were methionine,tyrosine,valine,ornithine,glutamine,serine,threonine,free carnitine,acetyl carnitine,propionyl carnitine,etc.Among the above metabolites,compared with the cold-damp block syndrome group,there were4 kinds of blood metabolites(1 kind of amino acid and 3 kinds of carnitine)with statistical difference between the poison-stagnant jaundice syndrome group and the cold-damp block syndrome group,which were ornithine,acetyl carnitine,propionyl carnitine,3-hydroxyoctadecarbonyl carnitine,etc.In the urine samples,compared with the normal control group,there were 11 kinds of urine metabolites(organic acids)with statistical difference between severe ICH syndrome(cold-damp block syndrome and poison-stagnant jaundice syndrome)group and normal group,which were2-hydroxy-isobutyrate-2,3-methyl-glutarate-2,3-hydroxy-3-methyl-glutarate-3,etc.Among the above metabolites,compared with the cold-damp block syndrome group,no urine metabolites with statistical difference were found between the poison-stagnant jaundice syndrome group and the cold-damp block syndrome group.2.Objectivity indicator results of LDHJ improving cholestasis in animal experimentsIn the third part of this study,We found that compared with the normal control group,the levels of TBIL,DBIL,TBA,ALT,AST,GGT and ALP in young rat model of cholestasis established by Alpha-naphthylisothi(ANIT) were significantly increased(P < 0.01),and the abnormal pathological changes were observed in liver tissue.Compared with model group,the levels of TBIL,DBIL,ALT,AST,TBA,GGT and ALP in LDHJ group were lower(P<0.01),and liver pathology was improved.In addition,there were statistically significant differences in serum metabolite levels between model group,LDHJ group and normal control group(P<0.05).Compared with the normal control group,there were 30 kinds of blood metabolites(4 kinds of amino acids and 26 kinds of carnitine)with statistical difference between the model group and the normal group,which were alanine,arginine,ornithine,threonine,free carnitine,malonyl carnitine,3-hydroxybutyryl carnitine,etc.Among the above metabolites,compared with the model group,there were 17 kinds of blood metabolites(1kind of amino acid and 16 kinds of carnitine)with statistical difference between the rats in the LDHJ group and model group,which were ornithine,free carnitine,malonyl carnitine,3-hydroxy-butyryl carnitine,etc.By comparing the differential blood metabolites between rats in model group and infants in ICH with severe syndromes,We screened out 13 universal and different blood metabolites(2 amino acids and 11 carnitines),which were ornithine,threonine,free carnitine,3-hydroxy-butyryl carnitine,3-hydroxy-iso-valeryl carnitine,etc.Conclusion1.The metabonomics technique adopted in this study is helpful to the elaboration of the essence of TCM syndrome differentiation in clinical practice,and the exploration of drug action mechanism in experiment.2.The second part of this study regard the infants as the object.General information such as gender,birth condition,birth mode,feeding mode,age and weight did not affect the subsequent metabolomics analysis of the two syndromes in ICH with severe syndromes(cold-damp block syndrome and poison-stagnant jaundice syndrome).In terms of the duration of disease and the levels of TBIL,DBIL,ALT,AST,TBA,GGT and ALP,patients with poison-stagnant jaundice syndrome had a longer course of disease than those with cold-damp block syndrome,and the degree of cholestasis and liver injury were more serious.27 kinds of differential blood metabolites(7 kinds of amino acids and 20 kinds of carnitine)and 11 kinds of differential urine metabolites(11 kinds of organic acids)in ICH with severe syndrome were screened by LC-MS/MS and GC-MS,which might be objective biomarkers of ICH with severe syndrome.Among them,4 kinds of blood metabolites(1amino acid and 3 carnitines: ornithine,acetyl carnitine,propionyl carnitine,3-hydroxyoctadecarboxylcarnitine)were objective indicators of differentiation between cold-damp block syndrome and poison-stagnant jaundice syndrome.The different metabolites found above are helpful to distinguish and explain the different blood and urine metabolites spectrum characteristics of ICH with severe syndromes(cold-damp block syndrome and poison-stagnant jaundice syndrome)from the normal control group.The differential hematuric metabolites suggest that the occurrence and development of ICH with severe syndrome is related to the degradation of amino acids,oxidation of fatty acids and metabolic pathways of organic acids,and fatty acid oxidative metabolism disorder is the main direction of differentiation of TCM syndrome types of this disease(cold-damp block syndrome and poison-stagnant jaundice syndrome).3.The third part of this study took young rats as the object.According to the pathological changes of liver and the levels of TBIL,DBIL,ALT,AST,TBA,GGT and ALP,serious liver function damage and cholestasis occurred in young rats with intrahepatic cholestasis induced by ANIT,indicating successful modeling.The liver pathology of LDHJ group improved cholestasis and the degree of liver function injury were also significantly reduced,indicating that LDHJ can improve the degree of cholestasis and liver injury.LC-MS/MS technique was used to verify the difference in blood metabolites between young cholestasis rats and normal young rats.LDHJ can reverse the levels of 17 different metabolites(1 amino acid and 16 carnitine)in young cholestasis rats and improve cholestasis,and the mechanism of action may be mainly regulating fatty acid oxidation metabolism. |
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