Study On The Mechanisms Of Asarum In Pulmonary-liver-kidney Toxicity Based On Network Pharmacology And Metabonomics Of ~1H NMR

ObjectiveNetwork pharmacology analysis is applied to forecast the active compounds of Asarum and the toxic targerts of Asarum in Lung-Liver-Kidney.The methods of metabonomics and network analysis of differential metabolites and the targets of toxicity are used to speculate the core mechanism with Molecular biology method to confirm.MethodsThe network pharmacology method was used to analyze and screen the active compounds and the toxic mechansims of Asarum in Lung-Liver-Kidney.The network of compounds-targets was constructed,Gene Ontology(GO)enrichment and pathway were analyzed to explore the material basis and possible toxic mechansims of Asarum in Lung-Liver-KidneyMale Sprauge-Dawley(SD)rats were randomly divided into 4 groups,high-dose group of Asarum(1.35 g/kg),medium-dose group of Asarum(0.81g/kg),low-dose group of Asarum(0.27 g/kg)and the control group.Drugs were gavaged for 28 days,The control group was given the same volume of distilled water.Tissue samples of Alveolar lavage fluid,lung,liver and kidney were collected on the 29^th day and subsequently analyzed by ~1H NMR after pretreatment.Lung toxicity screening of Asarum active compounds targets with high dose group differences in lung tissue metabolites,Asarum active compounds hepatotoxicity targets with high dose group of liver tissue differences metabolites,Asarum active compounds renal toxicity targets with high doses of asarum group differences metabolites,active ingredient lungs hepatorenal toxicity common targets with high dose group of lungs hepatorenal Asarum common differences metabolites.The key mechanism targets were screened by network interaction analysis based on Metaboanalyst,and the key mechanism targets were verified by imprinting hybridization of western-blot.Results192 kinds of compounds of Asarum were obtained by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).9kinds of potential active compounds were screened with thesholds of Oral bioavailability(OB)?30%and Drug-likeness(DL)?0.13.179 kinds of candidate targets of Asarum were obtained basing on the database of SWISS Target Prediction.From Gene Cards database,9188 kinds of targets of lung injury were obtained,and 815 kinds of candidate targets of lung toxicity were screened.8447 kinds of targets of liver injury were obtained,621 kinds of candidate targets of hepatotoxicity were screened,and 9078 kinds of targets of kidney injury were obtained,596 kinds of candidate targets of nephrotoxicity were screened.The potential active compounds of Asarum had 36 intersection targets with candidate targets of lung toxicity,29 intersection targets with candidate targets of hepatotoxicity,23 intersection targets with candidate targets of nephrotoxicity,and 14 intersection targets with candidate targets of Lung-Liver-Kidney toxicity.Network alalysis between the compounds and toxicity targets showed that the top three important compounds were SCHEMBL119969,kamanol and picrasidine D.GO analysis showed that the toxic mechanism of Asarum in Lung-Liver-Kidney mainly affected the biological process of cells.Pathway analysis showed that the toxicity of Asarum was closely related to tumor-related pathway,metabolism-related pathway,neurotransmitter related pathway and inflammatory related pathway.The common core targets of toxic mechanism of Asarum in Lung-Liver-Kidney were ACE,MAPK8,MTOR,PLG,PTGS2,PPARG,MMP9,ERBB2,GRIN2B,DRD2,COMT.Potential biomarkers of Asarum in lung toxicity were glucose,pyruvate,lactic acid,leucine,isoleucine,valine,proline,methionine,malic acid,creatine,cysteine,ethanolamine,choline,phospholipid choline,taurine,glycine and tryptophan.Asarum hepatotoxicity biomarkers were leucine,alanine,acetic acid,methionine,glutamic acid,aspartic acid,creatine,phosphatidyl choline,betaine,taurine,tryptophan,sugar,glucose.The renal toxic biomarkers of asarum were isoleucine,leucine,lysine,alanine,acetic acid,proline,methionine,glutamate,threonine,triethylamine,creatine,choline,phosphatidyl choline,betaine,taurine,inositol,glycine,tyrosine,phenylalanine and glucose.The toxic metabolism of Asarum in Lung-Liver-Kidney is mainly manifested in promoting amino acid metabolism,energy metabolism and lipid metabolism,increasing oxidative damage and magnifying inflammatory effects,resulting in cytotoxicity.Pathway analysis showed that there were three common metabolic pathways of glutamine and glutamate metabolism,taurine and taurine metabolism,glycine serine and threonine metabolism.Network analysis between differential metabolites and predicted targets network with mechanism verification:There were 18 kinds of differential metabolites in the lung tissues of the high-dose Asarum group,which were network analyzed with the potential targets.Two subset networks were obtained.Glutamate had the highest correlation with the main targets of ARG1,GRIN2B and ACE.Two subset networks were obtained by network analyzing of liver tissues.The most important substance was glutamate,involving targets of GRIN2B,APP,ARG1,DRD2 and COMT.Only one subset network was obtained by network analyzing of kidney tissues.The substances with high centrality greater were glutamate,lysine,and glycine,among which glutamate had the highest correlation,involving targets of ARG1,GRIN2B,ACE,APP,DRD2,COMT,PLG,and AGTR1.There were 6 common differential metabolites in the high dose Asarum group of Lung-Liver-Kidney tissues,which were analyzed with the potential targets.Only one subset network was obtained.The only important substance was glutamate,involving targets of GRIN2B,ARG1,DRD2,COMT.Glutamate metabolism may be the common mechanism of toxicity in the different tissues.Based on the experimental results and literature review,the targets of ABAT and GRIN2B related to glutamate metabolism were verified.Compared with the control group,the expression levels of ABAT and GRIN2B targets in Lung-Liver-Kidney tissues of high-dose Asarum group were significantly increased.ConclusionThe potential active compounds of Asarum were picrasidine D,caribine,cryptopine,sesamin,SCHEMBL119969,3-O-methylviolanone,kaempferol,asarinin and Patchoulol.The common potential biomarkers of toxicity of Asarum in Lung-Liver-Kidney were leucine,methionine,glutamate,creatinine,phosphatidyl choline and glucose.The toxic mechansims of Asarum in Lung-Liver-Kidney are closely related to energy metabolism,amino acid metabolism and lipid metabolism.The toxic mechansims of Asarum in Lung-Liver-Kidney was closely related to glutamate metabolism pathway.The protein expression of ABAT and GRIN2B,and the concentration of glutamate increasing in Lung-Liver-Kidney tissues of high-dose asarum group,suggested that asarum may cause neuroexcitatory toxicity.