| Purpose:Based on the untargeted metabolomics technology of gas chromatography-mass spectro metry(GC-MS),through urine research Qingshen granules treatment dampness-heat syndrome ofof chronic renal failure(CRF)of the syndrome of metabolic material basis and related metabolic pathways,and combined with the network pharmacology research,to provide scientific basis for clarifying the efficent substances and mechanism of action of Qingshen granules in the treatment of CRF damp-heat syndrome.Method:1.Clinical study: Morning urine was collected from 5 patients with CRF damp-heat syndrome and 5 healthy normal subjects respectively,and samples were obtained by low-temperature and high-speed centrifuge method.After sample pretreatment,non-targeted metabolomics method using GC-MS technology was used to obtain and pretreat metabolomics data.Then,multivariate statistical analysis was performed,and unsupervised principal component analysis(PCA)was used to study the overall distribution characteristics of the sample interval and evaluate the stability of the entire analysis process.Supervised partial least squares(PLS-DA)and orthogonal partial least squares(OPLS-DA)were then used to compare the overall differences in metabolic profiles between the groups to identify the differences in metabolites between the groups.The variable weight value(VIP)was obtained by OPLS-DA method,VIP>1.0was selected,and the differential metabolites with P<0.05 were T-tested.The enrichment of metabolic channels was studied using KEGG database.2.Experimental study: 40 2-month-old male SPF SD rats were fed adaptively for 1week and randomly divided into the normal group(10 rats)and the modeling group(30rats)according to 1:3.After 4 weeks of adenine intragastric administration,the rats were randomly divided into model group and Qingshen granule group,with 15 rats in each group.After 12 weeks of administration of Qingshen granules,10 samples were sent to each group for examination.24 h urine was collected by a clean metabolic cage 12 h after the last administration for metabolomics study.The next day,abdominal aorta blood samples were collected from the three groups of rats,and samples were collected from bilateral kidneys to measure serum creatinine(Scr)and urea nitrogen(BUN),and HE and Masson staining techniques were used to determine the changes in renal histopathology.The use of non-targeted metabolomics methods of GC-MS,combined with multivariate statistical methods such as principal component analysis,partial least squares discriminant analysis and orthogonal partial least squares analysis,screening VIP>1.0,and T test P<0.05 metabolites,compare normal group,model group,Qingshen granule group rat urine metabolic spectrum,analysis of metabolites in different groups.And KEGG database was used for enrichment analysis of metabolic pathway,to study the anti-renal fibrosis metabolic pathway of Qingshen granules.3.Combination analysis of metabonomics and network pharmacology: Combined with previous studies on the network pharmacology of Qingshen granules,the Venn diagram was used to integrate the network pharmacology and metabonomics KEGG pathway enrichment analysis data.Results:1.Clinical research results: A total of 168 different metabolites were obtained by urine comparison and screening between patients with CRF dampt-heat syndrome and healthy normal subjects.67 different metabolites were obtained after identification and comparison by KEGG,HMDB and other databases,mainly organic acids and organic oxides.The obtained differential metabolites were enriched by KEGG pathway analysis,and the screening criteria was P<0.05.A total of 38 meaningful KEGG pathways were obtained.The top 10 in order of P value from small to large were central carbon metabolism,protein digestion and absorption,aminoyl t RNA biosynthesis,ATP binding box transporter,mineral absorption,synaptic vesicular cycle,D-amino acid metabolism,alanine,aspartic acid and glutamic acid metabolism,glyoxalic acid and dicarboxylic acid metabolism in cancer,respectively.Glycine,serine and threonine metabolism.2 Experimental research results:2.1 Scr and BUN: Compared with normal group,Scr and BUN levels of rats in model group and Qingshen granule group were significantly increased(P< 0.05).Compared with model group,Scr and BUN in Qingshen granule group were significantly decreased(P < 0.05).2.2 Renal pathology: The structure of glomeruli,renal tubules and renal interstitium in the normal group was normal,while the model group showed mesangial proliferation,segmental sclerosis,local tubular atrophy,compensatory tubular hypertrophy,and large area of blue-stained collagen fibers in the renal interstitium.Compared with the model group,the renal pathological damage was alleviated in the Qingshen granule group.2.3 Metabolomics : Under the conditions of VIP value >1 and P<0.05,103 differential metabolites were obtained between the screening model group and the normal group,and 73 differential metabolites were identified by searching and comparing in KEGG and HMDB databases.Then,KEGG pathway enrichment analysis was performed on the results of differential metabolites analysis,and 28 meaningful KEGG pathways were obtained(P<0.05).The same method was used to screen Qingshen granule group and model group,45 differential metabolites were obtained,25 differential metabolites were identified,and 23 meaningful KEGG pathways were obtained(P<0.05).The intersection of the differential metabolites obtained from the two parts was used to obtain 8differential metabolites,which were fumaric acid,malic acid,n-acetyl-L-glutamic acid,L-serine,L-threonine,uracil,xanthuric acid and glutaric acid.The contents of these differential metabolites were down-regulated in the model group,up-regulated in the Qingshen granules group and tended to be normal.By intersecting the KEGG pathways obtained from different groups,14 common KEGG pathways were obtained,including central carbon metabolism,arginine biosynthesis,aminoacyl t RNA biosynthesis,and alanine,respectively.Aspartate and glutamic acid metabolism,glyoxylate and dicarboxylate metabolism,renal cell carcinoma,citric acid cycle(TCA cycle),glucagon signaling pathway,protein digestion and absorption,gamma-aminobutyric synapses,ATP binding box transporters,valine,leucine and isoleucine biosynthesis,pyrimidine metabolism,and tyrosine metabolism.3.Analysis results of metabonomics combined with network pharmacology Referring to the network pharmacological study of Qingshen Granules in the treatment of damp-heat syndrome of CRF completed by our research group,a total of 147 pathways were obtained through enrichment analysis of KEGG pathway with P<0.05 as the screening criterion.By integrating the KEGG pathway enrichment analysis data of network pharmacology and metabolomics,four pathways were finally obtained,including central carbon metabolism,protein digestion and absorption,ATP-binding box transporter,and renal cell carcinoma in cancer.Conclusions:1.Based on the GC-MS urine metabolomics studies,it is found that there are 8 potential biomarkers of Qingshen granules for the treatment of CRF damp-heat syndrome,including fumaric acid,malic acid,N-acetyl-L-glutamic acid,L-serine,L-threonine,uracil,xanthuric acid and glutaric acid.2.The metabolomics combined with network pharmacology analysis showed that the mechanism of Qingshen Granules in the treatment of CRF damp-heat syndrome is related to the regulation of central carbon metabolism in cancer,protein digestion and absorption.3.Qingshen Granules regulate amino acid metabolism,which is one of the important targets to delay the progression of renal fibrosis. |
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