| BackgroundStable coronary artery disease is a common chronic disease that threatens the quality of life and long-term prognosis of patients.At present,the management of stable coronary artery disease is mainly based on lifestyle intervention,intensified drug treatment and reduction of risk factors.But even after effective management,some patients still experience symptoms of myocardial ischemia.Percutaneous coronary intervention can reduce the number of angina pectoris,but it can cause a 5%to 10%risk of restenosis,and it is difficult to improve the long-term cardiovascular prognosis.The above problems indicate that new and individualized treatment strategies need to be developed for coronary artery disease to improve the efficacy and prognosis.Traditional Chinese medicine(TCM)treatment has unique advantages in preventing and treating coronary artery disease and improving the quality of life of patients through individualized treatment.Intermingled phlegm and blood stasis syndrome and blood stasis syndrome are the most common TCM syndromes of coronary artery disease.The basic researches on intermingled phlegm and blood stasis syndrome and blood stasis syndrome have have done a lot of work,but there are still some problems.Proteomics and TCM syndromes have some similarities.Both are the reflection of the body’s immediate functional state.Proteomics studies are equivalent to exploring the microscopic material basis of syndromes.In view of this,this study uses quantitative proteomics technology and bioinformatics methods to initially screen the plasma protein expression profiles of intermingled phlegm and blood stasis syndrome,and blood stasis syndrome of stable coronary artery disease,hoping to clarify the core biological process of the syndromes and the molecular biological basis of the phenotypic differences between two syndromes,so as to provide a basis for the objective research of TCM syndromes and the individualized treatment of coronary artery disease.ObjectivePreliminarily screen the plasma protein expression profiles of intermingled phlegm and blood stasis syndrome and blood stasis syndrome,explore the core targets and pathways in the biological process of intermingled phlegm and blood stasis syndrome and blood stasis syndrome,and explore the molecular biological basis of the differences between the two syndromes,providing a basis for the objective research of TCM syndromes.Method1.DIA-based protein sequencing of coronary artery disease intermingled phlegm and blood stasis syndrome and blood stasis syndrome.According to the"Guidelines for the Diagnosis and Treatment of Stable Coronary Artery Disease" and"Diagnostic Criteria for Elements of Coronary Artery Disease and Angina Pectoris",a total of 15 subjects were included in the study,divided into coronary artery disease intermingled phlegm and blood stasis syndrome group,coronary artery disease blood stasis syndrome group,and healthy controls group.After obtaining the subject’s informed consent,the fasting plasma of the subject was collected in the early morning and centrifuged to obtain a protein mixture.After protein extraction,enzymatic hydrolysis,and desalination,it is separated by liquid chromatography and identified by mass spectrometry.Using the DIA mode to obtain complete peptide information.Spectronaut Pulsar software was used to search and identify the original quantitative protein data.After preprocessing the original data,statistical analysis was performed in Excel,and the fold change and Rvalue was used to screen the difference proteins between different groups.The Ggplot2 package of R software was uesd to draw a volcano map for visual display.2.Analysis of function of differential protein between intermingled phlegm and blood stasis syndrome,and blood stasis syndrome of stable coronary artery disease.Bioinformatics analysis was based on the differential protein results in the first part:①GO enrichment analysis:use the David database to analyze the enrichment of the genes corresponding to the differential proteins in each group,Select items which had more than 3 enriched genes for display,and use The GOplot package of R software to draw chord diagrams.②KEGG signal pathway query:Use KEGG Mapper to query the differential genes that have been enriched in GO analysis,enter Uniprot ID into KEGG Mapper to query related signal pathway maps,and select signal pathway maps with more than 3 enriched genes for display.③Protein-protein interaction analysis:Use the String database to search the interaction network of different proteins,use Cytoscape software to optimize the protein interaction network,and obtain the core protein by calculating the number of nodes.Results1.When the fold change is 1.5,compared with the control group,19 proteins are down-regulated and 9 proteins are up-regulated in the coronary heart disease with intermingled phlegm and blood stasis syndrome.The up-regulated proteins include:Immunoglobulin kappa variable 3D-15,Immunoglobulin heavy variable 3-43,von Willebrand factor,Peroxiredoxin-2,Immunoglobulin heavy variable 6-1,Mannosyl-oligosaccharide 1,2-alpha-mannosidase IA,Immunoglobulin heavy variable 1-45,Immunoglobulin heavy variable 3-9,Protein S100-A9.The down-regulated proteins include:Insulin-like growth factor I,Platelet glycoprotein lb alpha chain,Immunoglobulin heavy variable 1-69,Immunoglobulin kappa variable 1-8.Dipeptidyl peptidase 4,Albumin,Plasma serine protease inhibitor,Complement factor H-related protein 2,Insulin-like growth factor-binding protein 3,Apolipoprotein C-I,Transferrin receptor protein 1,Insulin-like growth factor-binding protein complex acid labile subunit,Complement factor H-related protein 4,Extracellular matrix protein 1,Complement factor H-related protein 1,Transforming growth factor-beta-induced protein ig-h3,L-selectin,Complement factor H,Insulin-like growth factor II.Compared with the control group,22 proteins are down-regulated and 9 proteins are up-regulated in the coronary heart disease with blood stasis syndrome.The up-regulated proteins include:Immunoglobulin lambda variable 3-25,Immunoglobulin heavy variable 3-23,Immunoglobulin heavy variable 3-43,Immunoglobulin heavy constant alpha 2,Immunoglobulin heavy variable 3-49,C-reactive protein,Immunoglobulin lambda variable 6-57,Immunoglobulin heavy variable 3-9,Insulin-like growth factor-binding protein 2.The down-regulated proteins include:Immunoglobulin kappa variable 1D-13,Immunoglobulin lambda-like polypeptide 1,Multimerin-2,Roundabout homolog 4,Plasma serine protease inhibitor,Insulin-like growth factor-binding protein complex acid labile subunit,SPARC-like protein 1,Insulin-like growth factor-binding protein 3,Multivesicular body subunit 12B,Albumin,L-selectin,Complement factor H,Complement factor H-related protein 1,Platelet glycoprotein Ib alpha chain,A disintegrin and metalloproteinase with thrombospondin motifs 13,Intercellular adhesion molecule 2,Transthyretin,Gelsolin,Apolipoprotein L1,Vitamin D-binding protein,Extracellular matrix protein 1,Phospholipid transfer protein.Compared with the blood stasis syndrome,6 proteins are down-regulated and 8 proteins are up-regulated in the coronary heart disease with intermingled phlegm and blood stasis syndrome.The up-regulated proteins include:IgGFc-binding protein,Mannosyl-oligosaccharide 1,2-alpha-mannosidase IA,Desmoglein-2,Protein S100-A9,Haptoglobin-related protein,Multimerin-2.The down-regulated proteins include:I Dipeptidyl peptidase 4,Thrombospondin-1,Probable non-functional immunoglobulin kappa variable 3-7,Insulin-like growth factor-binding protein 2,Platelet factor 4,Immunoglobulin heavy variable 3-49,Immunoglobulin kappa variable 2-40,Aminopeptidase N.2.①The core biological process of coronary heart disease with blood stasis syndrome is the complement activation and chronic inflammatory response.The core signaling pathway is the pathway of the complement and coagulation cascade.The core targets are von Willebrand factor and complement factor H.②The core biological process of coronary heart disease with intermingled phlegm and blood stasis syndrome is excessive activation of platelets and abnormal cell protein metabolism.The core signal pathway is the complement and coagulation cascade,and growth hormone synthesis,secretion and action.The core targets are von Willebrand factor,complement factor H,insulin-like growth factor 1,insulin-like growth factor 2,insulin-like growth factor binding protein 3.③The biological basis and related molecular targets of the phenotypic difference between two syndromes are not clear,and may be related to inflammation and immune response.Conclusion①The potential biomarker of blood stasis syndrome of coronary arterial disease is complement factor H.The formation of blood stasis syndrome of coronary arterial disease is related to the process of complement activation and the regulation of immune response,and is related to the coagulation cascade reaction;Potential biomarkers are Von Willebrand factor,insulin-like growth factor-1,insulin-like growth factor binding protein-3;③The plasma proteomics and biological process of coronary arterial disease blood stasis syndrome and coronary arterial disease phlegm and blood stasis mutual syndrome are similar. |
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