Treatment Effects Of Pulsatilla Decoction In Dampness-heat Diarrhea By Metabolomics And Network Pharmacology Approach

Dampness-heat diarrhea(DHD)is a common disease in the clinical application of Chinese medicine.Pulsatilla decoction(PD)is a classic prescription for the clinical treatment of dampness and heat in traditional Chinese medicine,and it has good therapeutic effect on the diarrhea of dampness-heat ulcerative colitis.In order to study the effect mechanisms of PD on the treatment of dampness-heat diarrhea,this study established the rat model of dampness-heat diarrhea by simulating the complicated pathogenic factors,and the treatment was conducted with PD.By clinical pathology indexes to evaluate the treatment effect,based on urine metabolomics to investigate the metabolic system changes of urine at different stages of dampness-heat rats,further by serum and urine metabolomics method to explore the effects of PD on endogenous metabolites and metabolic pathways in dampness-heat diarrhea model rats,combined with network pharmacology to intestigate the "ingredients-targets-pathways" effects mechanism of PD on dampness and heat diarrhea.Methods:32 Wistar rats were randomly divided into 4 groups,male and female half:control group,model group,self-healing group,and PD group.Rat model was established with high sugar,high fat diet,high temperature and humidity environment and colibacillus injection.After the model was established,PD was given on the PD group.The clinical symptoms,signs and pathological changes of rats were observed during the experiment.The blood routine indexes of rats and the levels of serum pro-inflammatory cytokines were detected.Collected the urine samples of dampness-heat diarrhea model rats at the high sugar-high fat,high temperature and high humidity,intraperitoneal injection of e.coli stages,using UPLC-Q/TOF-MS/MS detection of metabonomics technology,the Progenesis QI software was used to preprocess metabolomics data,and the MetaboAnalyst multivariate statistical analysis was used to analyze data.Screening the urine different metabolites between the control group and the dampness-heat diarrhea model group at different stages based on FC>1.2 or<0.8,q-value<0.05,and VIP>1.0,through the HMDB database,heat map clustering analysis and Venny analysis,the common difference metabolites at different stages were obtained.The metabolic pathway and relative content of metabolites were analyzed.The serum and urine of rats in each group were analyzed,and the difference metabolites and target metabolic pathways were screened for the treatment effects of PD on dampness-heat diarrhea.Based on the pharmacological analysis platform of Chinese medicine system,the main active components,targets and related diseases were screened.Analysis platform system based on traditional Chinese medicine pharmacology screening of PD of the main active ingredient,targets and related diseases,by Cytoscape software build component-targets-disease direct action network,String construction of protein and protein interaction network,and by David software to analyze gene ontology(GO)and pathway enrichment analysis(KEGG).Results:(1)The clinical symptoms and signs of rats in dampness-heat diarrhea model group were consistent with the clinical manifestations of dampness-heat diarrhea,and the body appeared white blood cells,red blood cells,hemoglobin in rats,the number of neutrophils and monocytes number,number of lymphocytes and red blood cells deposited,tumor necrosis factor alpha and interleukin(IL)-1,IL-2 and IL-6 significantly higher(P<0.05),and platelet number decreased significantly and the intestinal inflammatory cell infiltration,mucosal epithelial tissue damage and other pathological changes.PD can improve the clinical symptoms and signs,the normal blood routine indexes and serum pro-inflammatory cytokine level and relieve the pathological changes of intestinal tissue.(2)Dampness-heat diarrhea model rats at the high sugar and high fat,high temperature and high humidity and intraperitoneal injection of e.coli stages have seven common urine metabolites,respectively,5-hydroxy-N-formylkynurenine,gamma-L-glutamyl-L-cysteine,L-formylkynurenine,N-acetyl-5-methoxytryptamine,linoleic acid,riboflavin-5-phosphate and L-tryptophan,mainly involving the metabolism of riboflavin,tryptophan metabolism and linoleic acid metabolism pathways.High sugar and high fat phase:the relative contents of 5-hydroxy-N-formylkynurenine,linoleic acid,and N-acetyl-5-methoxytryptamine were decreased significantly,gamma-L-glutamyl-L-cysteine,riboflavin-5-phosphate and L-tryptophan were increased significantly;High temperature and high humidity stage:the contents of L-formylkynurenine and linoleic acid increased significantly,while riboflavin-5-phosphoric and gamma-L-glutamyl-L-cysteine were decreased significantly.Intraperitoneal injection of e.coli stage:the contents of L-formylkynurenine,linoleic acid and L-tryptophan were significantly decreased,and gamma-L-glutamyl-L-cysteine and rib ofl avin-5-phosphori c were increased significantly.(3)17 potential metabolic markers(8 serum,9 urine)from serum and urine samples were screened,these markers may be associated with dampness-haet diarrhea mechanism of PD,serum biomarkers have been verified by reference substance metabolism of serum potential markers for PD intervention dampness-heat diarrhea,including 7-ketone deoxycholic acid,serotonin and trauma acid.compared with control group,the relative contents of 7-ketone deoxycholic acid and trauma acid significantly increased in damp-heat diarrhea model group rats(P<0.05),the relative content of serotonin significantly decreased(P<0.05),after treatment with PD,the relative contents of these markers callback,and close to the control group.Through the analysis of its related metabolic pathways,results showed that the glycerol phospholipid metabolism,tryptophan metabolism,GSH metabolism and the pentose phosphate pathway metabolic pathways,comprehensive analysis of serum and urine related metabolic pathways that PD play eff-icacy effects by the body metabolism regulating glycerol phospholipid metabolism,tryptophan metabolism,GSH metabolism and other metabolisms.(4)there were 45 important chemical components in the PD,and there were 166 targets and 343 related diseases."Ingredients-targets-disease" network analysis showed that PD mainly through the change of quercetin,β-sitosterol and stigmasterol chemical composition,roling in key targets TP53,JUN,IL6,TNF,VEGFA,FOS,EGF and MAPK1 to develop the eff-icacy in a variety of diseases.GO annotation and KEGG pathway enrichment analysis showed that PD through regulating MAPK1,FOS,CCND1,JUN,BAX,BCL2,GSK3B TP53,MYC and TGFB1 targets might play regulate roles on colorectal cancer,and IL-6,TNF,IL-4 JUN,RELA,IFNQ IL-1,STAT1,IL-10,TGFB1,and IL-2 targets might play regulate roles on inflammatory bowel disease pathways.It is shown that PD can play a role in the treatment of diseases through the mechanism of "multi-component －multi-target-multi-pathway".Conclusion:(1)PD play a pharmacodynamic role which may be through the anti-inflammatory,enhance immune cell function and relieve and protect intestinal tissue injury to treat dampness-heat diarrhea.(2)The pathogenesis of dampness-heat diarrhea organisms may be 5-hydroxy-N-formyl-kynurenine,L-glutamyl-L-cysteine,L-formyl-kynurenine,linoleic acid,melatonin,riboflavin-5-phosphate in metabolite markers affect riboflavin metabolism,tryptophan metabolism,and linoleic acid metabolic pathways,causing metabolic disorders in the body.(3)The effect mechanism of PD in treating dampness-heat diarrhea is through regulating the contents of serum biomarkers 7-ketone deoxycholic acid,serotonin and trauma acid in dampness-heat diarrhea.Regulating serum glycerophospholipid metabolism,glyceride metabolism,and tryptophan metabolic pathways.(4)PD may regulate the content of potential urine metabolites in the dampness-heat diarrhea and regulate glutathione metabolism,tryptophan metabolism,pentose phosphate pathway and biotin metabolism pathway.(5)PD can act on key target which are TP53,JUN,IL6,TNF,FOS,IFNQ IL-β,IL-1α,IL-2 and MAPK1 through active ingredients such as quercetin,β-sitosterol and stigmasterol,in order to regulate colorectal cancer pathways and inflammatory bowel disease pathways,the efficacy of curing dampness-heat diarrhea was laid,which laid a theoretical foundation for revealing the related biological effects of "multi-component-multi-target-multiple pathway-multiple disease" of PD.