Study On The Active Ingredients And Mechanism Of Guanxin Shutong Capsule In The Treatment Of Coronary Heart Disease Based On Network Pharmacology And Metabolomics

Objective:Coronary atherosclerotic heart disease(CHD)is a complex metabolic disorder disease with high morbidity and mortality.Guanxin shutong capsule(GXSTC)is a traditional Chinese medicine prescription based on the unique theory of Mongolian medicine.It has been used to treat CHD in clinic for many years,but its active ingredients and mechanism are still unclear.The purpose of this study is to establish a comprehensive research strategy combining with network pharmacology and metabolomics,to thoroughly study the active ingredients and mechanism of GXSTC in the treatment of CHD.Contents:This study was divided into two parts.The first one mainly included the construction of a molecular data set of GXSTC,gene ontology and kyoto encyclopedia of genes and genomes enrichment analyses,compound-target network and target-disease network establishment and analysis and molecular docking verification.The second one is to study the mechanism of GXSTC to treat CHD based on metabolomics,which mainly included the establishment of rat model of CHD,the determination of three important biochemical indicators and the detection of echocardiography.At last we used UHPLC-MS based untargeted metabolomics analysis method to identify the key biomarkers and metabolic pathways.Methods:1.Study on the mechanism of GXSTC in the treatment of CHD based on network pharmacology.Through a number of traditional Chinese medicine databases and literature mining,a molecular data set of guanxin shutong capsules was established.The candidate compounds were filtered according to the pharmacokinetic index requirements we set and related literatures.We used bioinformatics analysis tool to predict in vivo targets of candidate compounds and retain potential targets related to CHD then conducted pathway enrichment analyses including gene ontology and kyoto encyclopedia of genes and genomes.Cytoscape software(version 3.6.1)was used to construct the compound-target and target-disease networks to screen the active ingredients and targets of guanxin shoutong capsule.Finally,the molecular docking software Auto Dock(version 4.2.6)was used to conduct molecular docking to verify the predicted ingredients’ s activity.2.Study on mechanism of GXSTC in the treatment of CHD based onmetabolomics.A rat model of CHD was established by ligating the left anterior descending coronary artery,and the CHD model rats were divided into disease model group(Model,n = 6)and medication group(Treat,n = 6),the other rats without ligation were the sham operation group(Sham,n = 6).After four weeks of treatment with GXSTC,the rats in three groups were conducted detection of echocardiography and measurement of the contents of creatine kinase,lactate dehydrogenase and aspartate aminotransferase in the serum with biochemical kits.Then evans blue and2,3,5-triphenyltetrazolium chloride were used to prepare rat myocardial infarction sections.The metabolites in the plasma of three groups of rats were isolated and quantified by UHPLC-MS untargeted metabolomics.And the obtained original peak area data were analyzed by multivariate statistical method,then the differential metabolites between different groups were screened based on the set thresholds.The mz Cloud spectrum library,Thermo Scientific mz Vault spectrum library and human metabolism database were used to identify the different metabolites,and the biomarkers in the treatment of CHD were obtained.Finally,Metabo Analyst 4.0 was used to analyze the metabolic pathways of biomarkers.Results:1.The compound-target networks were successfully established,and 12 active ingredients with anti-inflammatory,anti-oxidative or anti-apoptotic activities in GXSTC were obtained by topology analysis.Moreover,we predicted four potential active compounds that had not been reported,and verified their anti-inflammatory activities using molecular docking.By establishing and analyzing the potential target-disease network of GXSTC,we found that it also has the potential to treat alzheimer’s disease.2.The rat model of CHD was successfully constructed.The experimental results showed that GXSTC could recover the increasing levels of creatine kinase,lactate dehydrogenase and glutamate transaminase,restore the damaged cardiac function of rats and significantly reduce the myocardial infarction area after ligation of the left anterior descending branch of coronary artery.We obtained 22 biomarkers and 9metabolic pathways significantly affected by GXSTC in the treatment of CHD and GXSTC could restore these disordered biomarkers and metabolic pathways.Phenylalanine metabolism,tryptophan metabolism,valine,leucine and isoleucine degradation,glycerophospholipid metabolism and phingolipid metabolism were the five most influential metabolic pathways,and they were associated with inflammation,lipid metabolism and oxidation,and apoptosis in the body,which was consistent with the results of network pharmacology that GXSTC had anti-inflammatory,antioxidant and anti-apoptotic activities.Conclusions:In this study,we established a comprehensive research strategy combining network pharmacology,molecular docking and untargeted metabolomics,to conduct systematic research on the active ingredients,in vivo targets and mechanism of GXSTC in the treatment of CHD.16 key ingredients with anti-inflammatory,antioxidant and anti-apoptotic activities in GXSTC were obtained,in which 4unreported compounds were predicted and their anti-inflammatory activities were verified by molecular docking.Moreover,we found that GXSTC had the potential of treating alzheimer’s disease.22 biomarkers and 9 metabolic pathways were obtained which were significantly affected by GXSTC in the treatment of CHD and GXSTC could restore these disordered biomarkers and metabolic pathways.All the above results indicated that the therapeutic mechanism of GXSTC on CHD is to reduce the inflammatory response in vivo,inhibit lipid peroxidation and myocardial cell apoptosis.