Clinical And Metabolomics Study On Spleen Deficiency Diarrhea In Children Treated By Warming And Moving Spleen-yang

ObjectiveBased on nearly 50 years of clinical practice,the academic viewpoint of treating children's spleen-deficency diarrhea mainly by warming and moving spleen Yang was put forward.The theoretical origin of the academic viewpoint was researched in this study.The characteristics of children's spleen-deficency diarrhea and the combined factors were explored and the medication rules and evaluate the clinical effect of tutor's treatment of children's spleen-deficency diarrhea were dug out.The metabolic profiles of urine and feces were analyzed in spleen-deficency diarrhea of children to speculate the possible mechanism of Wenyun granules.MethodsIn the first part of this study,the tracing of warming Yang therapy and moving spleen therapy is completed through the analysis of the theoretical basis,physiological and pathological basis,theories and development of TCM children's warming Yang therapy and moving spleen therapy.The theoretical connotation of Warming Yang moving spleen method is summarized through the sorting of the theoretical basis of syndrome differentiation and treatment of children's spleen-deficiency diarrhea.Three kinds of data mining techniques,frequency Statistics,association rules and cluster analysis,were adopted to summarize the outpatient cases and prescriptions of spleen deficiency diarrhea,to find out the combined or previous factors of spleen deficiency diarrhea.The core prescriptions and the addition and subtraction prescriptions for spleen-deficiency diarrhea were explored.In the second part of this study,urine samples of 30 children with diarrhea due to spleen deficiency were collected,and another 30 normal children were collected.Urine samples were detected by UHPLC-MS and GC-MS,and principal component analysis(PCA)and discriminant analysis of orthogonal partial least squares(OPLS-DA)were used to analyze the data,to screen the differential metabolites.The differential metabolites were identifiedin databases.The metabolic pathway involved in the differential metabolites in urine of children with spleen deficiency diarrhea and the metabolic pathway involved in the intervention of Wenyun granules were found.In the third part of this study,the fecal samples of children with spleen-deficiency diarrhea were collected from the clinic.GC-MS technology was used and PCA and OPLS-DA were used to analyze the data to screen the differential metabolites.The differential metabolites were identified in databases.Metaboanalyst 4.0 was used to find the metabolic pathways involved in the differential metabolites in the feces of children with spleen deficiency diarrhea and the metabolic pathways involved in the prognosis of the effective prescription Wenyun granules.In the fourth part of this study,Receiver Operating Curve(ROC)analysis was used to test the diagnostic ability of differential metabolites for spleen deficiency diarrhea and to help predict the target of Wenyun granules.ResultsThe first section of the first part of this study traces the origin of the methods of warming and moving spleen-yang in pediatrics of Chinese medicine.It is believed that the therapy of warming and moving spleen Yang is established on the physiological and pathological characteristics of children.Practitioner of Pediatrics in successive dynasties had played an important role in etiology recognition,health care and syndrome differentiation of spleen-deficency diarrhea.The second section show that diarrhea due to spleen deficiency in children mostly belongs to the category of protracted and chronic diarrhea in modern medicine from the perspective of the course of disease,and the age of onset is mainly infants,boys are more than girls,the season of onset is all year round,with more in summer,autumn and winter.The proportion of breast milk feeding for children with diarrhea due to spleen deficiency is relatively low.The etiology or combined factors of spleen-deficiency diarrhea in children,such as infection factors,lactose intolerance,food allergy or intolerance,antibiotics and allergic diseases,indirectly suggests that spleen asthenia diarrhea in children may be related to intestinal flora.The core prescription of tutor's treatment of spleen deficiency diarrhea in children is summarized through data mining.Wenyun granules consist of Cangzhu,Fuling,Paojiang,Yizhiren,Maiya and Sharen.The results of the second part of this study showed that there were 87 kinds of urine differential metabolites between children with spleen asthenia diarrhea and normal children,involving aminoacyl tRNA biosynthesis,D-glutamine and D-glutamic acid metabolism,glycine,serine and threonine metabolism,histidine metabolism,alanine,aspartic acid and glutamic acid metabolism,phenylalanine metabolism,glyoxylate and dicarboxylic acid metabolism and arginine biology,butyric acid metabolism,glutathione metabolism,and taurine and low calcium amino acid metabolism.Three metabolic pathways of glyoxylate and dicarboxylic acid metabolism,phenylalanine metabolism and TCA cycle were showed after treatment by Wenyun granule.The results of the third part of this study showed that there were 26 kinds of fecal differential metabolites between children with spleen asthenia diarrhea and normal children,involving the metabolism of arginine and proline,?-alanine metabolism,pyrimidine metabolism,glycine,serine and threonine metabolism,alanine,aspartic acid and glutamic acid metabolism,pantothenic acid and coenzyme A biosynthesis,D-glutamine and D-glutamic acid metabolism,nitrogen metabolism.In addition,the contents of acetic acid,propionic acid,butyric acid,isobutyric acid,valeric acid and isovaleric acid in feces were significantly lower than those in the control group.The different metabolites of faeces in children with diarrhea due to spleen deficiency were involved in the pathways of arginine and proline metabolism and glycine,serine and threonine metabolism after treatment by Wenyun granule.Wenyun granule can significantly improve the level of butyric acid,isobutyric acid and isovaleric acid in the intestine of children with spleen deficiency diarrhea.The results of the fourth part of this study show that the three different metabolites of cis-aconitic acid in urine,glutamine and guanidinoacetic acid in feces can distinguish children with spleen deficiency diarrhea from healthy children and the differences in metabolism before and after treatment of Wenyun granules.ConclusionThe academic viewpoint of treating children's spleen-deficency diarrhea mainly by warming and moving spleen Yang is based on theory and practice,which has clinical value.Spleen-deficiency diarrhea caused the metabolism disorder of carbohydrate,amino acid and intestinal flora in children.It is speculated that the damage and repair of intestinal mucosa,the imbalance of intestinal immunity and intestinal flora may be the important pathogenesis of spleen deficiency diarrhea.The possible mechanism of Wenyun granule is to improve the energy supply of intestinal mucosa metabolism by regulating the metabolism of carbohydrates and amino acids,and to promote the repair of mucosa,regulate the balance of intestinal flora and improve the internal environment of intestine.The three different metabolites of cis-aconitic acid in urine,glutamine and guanidinoacetic acid in feces may be helpful in the diagnosis of spleen deficiency and diarrhea and the target of Wenyun granules.We analyzed the differential metabolites of urine and feces in children with spleen deficiency diarrhea,and established a characteristic library based on the differences in the expression of potential biomarkers in urine and feces.Based on metabolomics,the scientific connotation of Wenyun granules in treating child diarrhea due to spleen deficiency was revealed.