| Purpose Based on the ultra-performance liquid chromatography-mass spectrometry(UPLC-MS)non-targeted metabolomics research method,the metabolite basis and related metabolic pathways of renal clear granules in the treatment of chronic renal failure damp fever were studied,and their effects on renal interstitial fibrosis were analyzed,and combined with network pharmacology related research,this paper provided a scientific basis for the treatment of chronic renal failure with kidney clear granules.Method1.Metabolomics1.1 Clinical study: 5 patients with CRF humid fever and 5 normal people,fasting elbow vein blood collection were divided into healthy control group and CRF damp-heat syndrome group,plasma metabolites were analyzed using High-performance liquid chromatography-mass spectrometry(UPLC-MS)non-targeted metabolomics methods,followed by univariate statistical analysis,multivariate statistical analysis,and differential metabolite screening,k EGG enrichment pathway was analyzed.1.2 Experimental study: adenine-induced renal fibrosis rats: 40 rats were fed adaptively for 1 week,and randomly divided into control group(Group C,N = 10)and model group(Group C,N = 30)according to 1:3 ratio [15].Adenine(2.5 G)was mixed with100 ml normal saline to form suspension.The suspension was given at a dose of 200 mg · kg-1 · d-1(0.8 ml · 100 g-1 · d-1)per day according to the body weight of rats.The control group was given the same amount of normal saline every day.After4 weeks,30 rats were randomly divided into model group(m group)and kidney-clearing group(Q Group),15 rats in each group.The nephrocleansing group was gavaged with 4.0g/kg aqueous solution of nephrocleansing granules,4ml once a day,and administered continuously for 12 weeks.Each group sent 10 rats for testing.Transabdominal aorta blood was collected,part of the blood sample was detected for Scr,BUN,and the remaining blood sample was used for metabolomics research.Renal tissues were collected for pathological examination.Plasma metabolites were analyzed using UPLC-MS non-targeted metabolomics methods,followed by univariate statistical analysis and multivariate statistical analysis to screen for differential metabolites,the key target of anti-renal fibrosis of cleansing granules was obtained;KEGG enrichment pathway analysis was performed.2.Metabonomics combined with network pharmacology2.1 Network pharmacology research,the Systems pharmacology of Qingshen granule was searched through TCMSP,encyclopedia of Traditional Chinese Medicine(ETCM),2015 Chinese Pharmacopoeia,cnki and Pubmed,objective: to construct the active component target of qingshen granule.2.2 By pairing the Qingshen granule with the gene set regulated by dampness-heat syndrome of CRF,we can get the genes which are affected by both the compound prescription and the disease,which may be the potential target of Qingshen granule to intervene the chronic kidney disease of dampness-heat syndrome of CRF.To construct the regulatory network of“Traditional Chinese medicine-active components-intersection target-disease”,and to analyze the intersection target by GO functional annotation and KEGG pathway enrichment.2.3 Metabonomics combined with network pharmacology in animal experiment,the differential metabolite names between model group and Qingshen Group were transformed into the ID of KEGG,which were introduced into METSCAP of Cytoscape to construct a metabolite-gene target network,166 gene targets were screened,and 106 crossing targets in network pharmacology were analyzed by PPI of STRING,then the two were integrated,and the important targets were selected by Degree value in network again.Result1.Metabolomics results1.1 Clinical research After identification,47 different metabolites were screened,mainly concentrated in lipids and lipid-like molecules,organic acids and derivatives,organic oxides and so on.It was enriched in choline metabolism,glycerophospholipid metabolism,glycine,serine and threonine metabolism,valine,leucine and L-Isoleucine biosynthesis in cancer(p <0.05);Choline metabolism in cancer was the most significant pathway(p < 0.01),involving two different metabolites,phosphatidylcholine(Ptd Cho C00157)and 1-acyl glycerophosphatecholine(1-acyl GPC C04230),involved in two signaling pathways,glycerophospholipid metabolism,MAPK signaling pathway.1.2 Experimental studies1.2.1 The rat model of renal fibrosis induced by adenine was established successfully.The experimental results showed that qingshen granule could decrease the content of SCR and BUN,alleviate the pathological damage of the kidney,and decrease the proliferation of collagen fibers in the renal interstitial.1.2.2 After searching,33 differential metabolites were screened from the control group and the model group,and 25 differential metabolites were screened from t he model group and the kidney-clearing group.Compared with the control group,the metabolites of the model group were mainly concentrated in lipids and lipid-like molecules,organic acids and derivatives,etc.,the differential metabolites w ere mainly concentrated in lipids and lipid-like molecules,and could down-regula te the up-regulated five differential metabolites in the model group:PC(16:0/18:3(9Z,12 Z,15Z))、PC(18:3(6Z,9Z,12Z)/20:3(8Z,11 Z,14Z))、PC(20:4(8Z,11 Z,14Z,17Z)/18:3(6Z,9Z,12Z))、Sphinganine、N2-Acetylornithine。1.2.3 After KEGG enrichment analysis,the control and model groups were enriched for significant signaling pathways such as choline metabolism,glycerin metabolism,sheath lipid metabolism,sheath lipid signaling pathway(p < 0.05).The model and clear kidney groups were enriched for significant signaling pathways such as choline metabolism,glycerin lipid metabolism,and amino t RNA biosynthesis in cancer(p < 0.05).In the two metabolic pathways of choline metabolism and glycerophospholipid metabolism in cancer,Qingshen granule can down-regulate the differential metabolite Ptd Cho after administration.2.Metabolomics and network pharmacology analysis of Qingshen granule2.1 There are 106 potential targets of Qingshen granule in network pharmacology for chronic kidney disease damp-heat syndrome.2.2 Network pharmacology: the CC enrichment in GO included plasma membrane,plasma membrane components,cytoplasm and so on.There were 167 KEGG enrichment pathways,including choline metabolism and MAPK signaling pathway in cancer.2.3 According to Venn diagram’s analysis,the intersecting pathway is choline metabolism in cancer.2.4 A metabolite-gene target network was constructed and 180 related genes were obtained.After PPI analysis,there were ACTB,Jun,PPARG,PTEN,ESR1,PLD2,PLA2G4 A,PLA2G1B,PIK3 CA,PLA2G6,CCL2,PRKCB and other important targets.Conclusion1.Metabolomics studies identified five potential biomarkers of Qingshen granules for the treatment of CRF wet heat syndrome,closely related to biomarker Ptd Cho,it isinvolved in Glycerophospholipid metabolism,choline metabolism in cancer and MAPK signal pathway.2.Combining metabonomics with network pharmacology,we conclude that Qingshen granule can inhibit renal fibrosis by regulating lipid metabolic disorder and reducing inflammatory reaction,the pathway of action may be closely related to choline metabolism in cancer and MAPK signal pathway. |
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