Study On The Clinical And Mechanism Of Metabonomics And The Intervention Of TCM Prescriptions For Clearing Method Of Damp-heat Syndrome Of Isolated Hematuria In Children

Objective:This study is based on metabolomics technology and conducts a prospective case-control study on clinical data of children with isolated hematuria(IH).It analyzes the clinical efficacy of using traditional Chinese medicine Clearing Method formula to intervene in children with hematuria,and reveals the mechanism of action of Jinshuiqing mixture(JSQ)in the treatment of dampness heat syndrome in children with hematuria at different levels such as clinical biochemistry,cytokines,and metabolomics;Exploring landmark metabolites in children with hematuria(dampness heat syndrome);And for the first time,we attempt to establish a warning model that combines multiple levels such as clinical biochemistry,cytokines,and metabolomics to assess the progression of hematuria disease.The aim is to provide objective evidence support for clinical doctors to choose the intervention time for hematuria children without conducting tissue invasive testing in clinical practice.Method:Part 1:Clinical and Mechanism Study on the Intervention of Traditional Chinese Medicine Clearing Method Formula in Children with Solitary HematuriaAll cases in this study were from hospitalized children in the Department of Pediatrics of Hubei Provincial Hospital of Traditional Chinese Medicine and the first visit of Professor Liu Xiaoying’s outpatient department from 2018 to 2021.After strict screening according to the inclusion and exclusion criteria,a total of 130 cases were included,including 29 cases normal control groups,49 cases in the damp heat syndrome group,and 52 cases in the non damp heat syndrome group.Organize,statistically analyze,and evaluate the clinical efficacy,long-term efficacy,onset time,degree of IH level change before and after treatment,safety,and adverse event outcomes of Jinshuiqing as the main treatment for children with hematuria;Changes in physical signs,biochemical indicators,and cytokines before and after treatment;Observe the impact of birth season and onset season on the incidence of hematuria;Observe the distribution of pathological types in hematuria renal biopsy,the correlation between urinary RBC and pathological severity,the correlation between pathological types and traditional Chinese medicine syndrome types,and the relationship between pathological severity and therapeutic effect.Part 2:Metabolomics study on the intervention of traditional Chinese medicine Clearing Method formula in isolated hematuria in childrenOn the basis of the first part,the collected urine samples were detected by nuclear magnetic resonance,pretreated by spectrogram,and relatively integrated.Then,the SIMCA software was used for pattern recognition multivariable analysis.The unsupervised principal component analysis PCA,supervised partial least squares discriminant analysis(PLS-DA),and orthogonal partial least squares discriminant analysis(OPLS-DA)were used for pairwise comparison to evaluate the model quality and prediction ability,and the replacement test was used to determine whether the model was overfitting,And combined with cross validation analysis of variance,the P-value size was used to test the effectiveness of the model.Use the four choice triple standard method to screen metabolites with significant differences and draw a volcanic map.Import potential differential metabolites into MetaboAnalyst online software,combine SMPDB and KEGG databases for enrichment analysis of metabolic pathways,and construct and draw metabolic pathway network diagrams.Part Ⅲ:Exploratory research on the risk early-warning model of children’s hematuria diseaseOn the basis of the aforementioned two parts,various indicators at different levels,including demographic characteristics,biochemical indicators,metabolomics,cytokines,and other indicators,are converted into WOE values.Then,IV screening,correlation screening,and coefficient screening are performed.Analysis is conducted using Python,and a multi-level warning model is constructed using Logistic Regression and BFGS algorithms to jointly judge the degree of progression of hematuria disease.Result:Part 1:Clinical and Mechanism Study on the Intervention of Traditional Chinese Medicine Clearing Method Formula in Children with Solitary Hematuria1.Demographic indicators:There were no statistically significant differences in gender,age,body mass,height,BMI values,etc.among the three groups.2.Physical signs and biochemical indicators:①Blood pressure:There was no statistically significant difference in overall systolic and diastolic blood pressure.The number of people with increased systolic blood pressure in the non damp heat syndrome group was statistically different from that in the damp heat syndrome and control group;②Serum creatinine:The control group,damp heat syndrome group,and non damp heat syndrome group are all within the normal range.Compared with the damp heat syndrome group,the non damp heat syndrome group has an increase in serum creatinine value ASO:Compared with the control group,the non damp heat syndrome group showed an increase.④C3 complement:Compared with the control group,both the damp heat syndrome and non damp heat syndrome groups showed a decrease,and the non damp heat syndrome group showed a more significant decrease.⑤Blood viscosity low shear 1(1/S):Compared with the control group,the non damp heat syndrome group increased.⑥ESR:Compared with the control group,the non damp heat syndrome group showed an increase with significant statistical differences.⑦ Vitamin D:Compared with the control group,both the damp heat syndrome group and the non damp heat syndrome group showed a significant increase,with a significant difference.3.Birth and onset season indicators:The Pearson chi square test results showed no statistical difference between the groups,indicating that the birth season had no significant impact on the incidence of IH.The frequency of summer onset in the dampness heat syndrome group is higher than that in the non dampness heat syndrome group,and there may be a certain trend in the onset seasons of different syndrome types.Among them,the dampness heat syndrome group is more common in summer and winter,while the non dampness heat syndrome group is more common in winter and spring.Moreover,compared with the non dampness heat syndrome group,the dampness heat syndrome group is more prone to frequent hematuria in summer.There was no statistical difference in the correlation between the birth season and the onset season.4.Hematuria observation indicators:①Course of disease:In the damp heat syndrome group,there are more children with IH in the early stage(within half a year of onset)than in the non damp heat syndrome group,and in the non damp heat syndrome group,there are more children with IH in the later stage(within one and a half years of onset)than in the damp heat syndrome group.②Safety and adverse event outcomes:There is no safety or adverse event outcome,and overall,the Clearing Method formula has good safety in treating this disease.5.Hematuria efficacy indicators:① Inter group comparison:There was no statistically significant difference in the onset time of JSQ intervention between the damp heat syndrome group and the non damp heat syndrome group.The damp heat syndrome group has a recent onset rate of 69.4%,a total effective rate of 83.4%,and a total effective rate of 95%in the long term.This indicates that the onset time is fast,the therapeutic effect is good,and the duration is relatively long Comparison before and after treatment:Most levels of urinary RBC before treatment are distributed in levels 1-3,and can be recalled to levels 0-2 after treatment,with significant statistical differences.Urine after treatment in the damp heat syndrome group β Indicators such as 2-MG,serum IgA,C3 complement,and low shear blood viscosity all showed corresponding reductions compared to before treatment.6.Cytokines:Compared with the control group,the NF of the damp heat syndrome group and the non damp heat syndrome group before treatment-κB and TGF-βThe expression levels of both groups significantly increased,and after treatment,the expression levels of both groups decreased to the level of the control group;The expression levels of HGF in both groups were upregulated after treatment compared to before treatment,and the dampness heat syndrome group and non dampness heat syndrome group were upregulated compared to the control group after treatment.7.Renal tissue pathology:There is no statistical difference between the severity of hematuria and the severity of pathology in each grading;There is no correlation between traditional Chinese medicine syndrome types and pathological diagnosis types;There is no correlation between pathological severity and short-term or long-term efficacy.Part 2:Metabolomics study on the intervention of traditional Chinese medicine Clearing Method formula in isolated hematuria in children1.Identify the possible differential peaks corresponding to the differential chemical shift points,and assign 75 metabolites and 11 unidentified metabolites from the spectrum.2.Multivariate analysis results:①PLS-DA:The control group,before/after treatment with damp heat syndrome,and before/after treatment with non damp heat syndrome showed an overall trend of separation among the five groups.Three groups were compared,and there was a significant difference between the damp heat syndrome group before and after treatment compared to the control group.After JSQ treatment,the distribution of samples was close to that of the control group;The overall difference between the non damp heat syndrome group before and after treatment and the control group is more significant than that of the damp heat syndrome group.After JSQ treatment,the distribution of samples is closer to that of the control group;The comparison between the dampness heat syndrome group,non dampness heat syndrome group,and control group can be distinguished,indicating that there are differences in metabolic levels between the dampness heat syndrome group and the non dampness heat syndrome group;The difference between the damp heat syndrome and non damp heat syndrome groups and the control group after the intervention of clearing methods is not significant,indicating that both groups have returned to the level of the control group after treatment OPLS-DA:There is a metabolic difference between the damp heat syndrome group and the control group.After treatment,the metabolic profile of the damp heat syndrome group and the non damp heat syndrome group can be recalled to the level of the control group;Compared with the non damp heat syndrome group,the classification effect of the model is good and there is a significant difference;After treatment,there was no statistical difference between the damp heat syndrome group and the non damp heat syndrome group,indicating that the two treatments were effective.3.Statistical information related to different metabolites screened from different groups:①Control group and damp heat syndrome group:2 metabolites were upregulated and 5 metabolites were downregulated.② Damp heat syndrome group and non damp heat syndrome group:2 metabolites were upregulated and 9 were downregulated.③ After the treatment of dampness heat syndrome,the group and control group showed upregulation of 3 metabolites and downregulation of 7 metabolites.④ Damp heat syndrome and non damp heat syndrome groups:1 metabolite expression is upregulated.4.Pathway enrichment results:The three groups of dampness heat syndrome group,dampness heat syndrome treated group,and non dampness heat syndrome treated group have statistically significant metabolic pathways that are the same as the control group,totaling 7 pathways,specifically:① citric acid cycle;②Amino sugar metabolism;③ Pyruvate metabolism;④ Glutamic acid metabolism;⑤ Metabolism of arginine and proline;⑥Aerobic glycolysis;⑦Glycine and serine metabolism,etc.Before treatment,the statistically significant metabolic pathway between damp heat syndrome and non damp heat syndrome is aspartate metabolism.After treatment,there was no difference in metabolic pathways between dampness heat syndrome and non dampness heat syndrome,indicating that the traditional Chinese medicine Clearing Method and Jinshuiqing were effective as the main pre treatment for this disease.Part Ⅲ:Exploratory research on the risk early-warning model of children’ s hematuria diseaseThe results showed that based on:Demographic characteristics:①Onset season,②Age;Biochemical indicators:③ blood viscosity,④Vitamin D;Cytokines:⑤TGF-β;Metabonomics:⑥Alanine,⑦Acetate.A regression equation can be established for a total of 7 joint indicators in 4 different dimensions,and further validation will be conducted using data from children undergoing renal biopsy as the validation set.It is found that when the predicted value of disease probability is greater than 60%,the prediction accuracy can reach 89.47%.Conclusion:This study uses Jinshuiqing mixture to treat isolated hematuria dampness heat syndrome in children,which has a fast onset time,good efficacy,and a long duration.There is no safety or adverse event outcome.The mechanism of treating isolated hematuria dampness heat syndrome in children with traditional Chinese medicine Qingfa formula and Jinshuiqing mixture as the main treatment may be:1.callback urine β 2-MG,complement C3,serum IgA,and TGF-β、NF-κB.The expression level of cytokines such as HGF;2.The target of action involves metabolic products closely related to hematuria dampness heat syndrome,as well as metabolic pathways such as glucose metabolism,amino acid metabolism,and gut microbiota;It can alleviate the pathway of renal injury mediated by inflammatory factor response,reverse the disturbance and imbalance of metabolic substances and pathways closely related to hematuria dampness heat syndrome,and maintain low levels of expression of red blood cells in the urine of children with hematuria,thereby protecting the kidney and delaying the chronic process of renal tissue.In addition,a preliminary risk warning model for children with hematuria was established,with a total of 7 combined indicators from four different dimensions of demographic characteristics,biochemical indicators,cytokines,and metabolomics.Regression equations were established and incorporated into the training set of children with renal biopsy IH to validate the model.The prediction accuracy was high,indicating that when there were changes in the combined indicators,it was suggested that children with hematuria may have had renal tissue pathological changes and timely intervention and treatment were needed.This provides a methodological approach for clinical physicians to alert children to the risk of hematuria,and takes a small step towards the practical application of non-invasive testing instead of renal biopsy in clinical practice.