| The target is the source of drug action which could be the most direct,important,and initial biological basis for disease treatment.The characteristic of traditional Chinese medicine is multi-component and multi-target.The complexity of multicomponents makes mechanism research challenging.The mechanism of traditional Chinese medicine is the change of the overall functional state of organisms through the direct or indirect action of drug molecules with the target proteins of single or multiple cells,which is the cascade reaction generated by the action of several key node proteins in the disease network.Therefore,it is particularly important to explore the direct targets of the active components.The magnetic bead "fishing" technique is an experimental technique for the direct action screening of the effective components.It has the advantages of speed,ease of operation,high sensitivity and specificity.The specific method is to label magnetic beads,take magnetic particles as the core and modifying them externally.The surface can be combined with different chemical functional groups,such as-NH2,-COOH,-NHS,etc.These groups can bond with the active groups of selected drug monomers to form magnetic bead-drug complex.The magnetic bead-drug-protein complex formed for fishing the target protein can bind to the drug monomer.After protein elution,the target protein is separated,and then the target protein is identified.Finally,the direct target of the active ingredients of traditional Chinese medicine can be obtained.Shuangshen Ningxin Capsule is a compound Chinese medicine capsule developed by the Institute of Basic Medical Sciences of Xiyuan Hospital,China Academy of Chinese Medical Sciences.It is composed of the effective parts of Ginseng,Salvia Miltiorrhiza and Rhizome Corydalis.In recent years,there have been many researches reported that Shuangshen Ningxin Capsule show significant efficacy in the treatment of coronary heart disease,angina pectoris,myocardial ischemia,coronary microvascular disease and other cardiovascular diseases.These experiments have proved that Shuangshen Ningxin Capsule has a good effect on the prevention and treatment of myocardial ischemia-reperfusion injury.However,the direct target has not been studied.Based on this,this study adopted magnetic bead "fishing" technology to explore the direct target of effective components of Shuangshen Ningxin Capsule in preventing myocardial ischemia reperfusion injury and conducted preliminary experimental verification.Objective:Via the magnetic bead "fishing" technology,the magnetic bead-Shuangshen Ningxin Capsule active component complex was synthesized and mixed with the protein solution to generate the magnetic bead-Shuangshen Ningxin Capsule active component-target protein complex.The target protein was eluted,separated,and identified by mass spectrometry.Then bioinformatics analysis,molecular butt coupling and other methods were used to screen out reliable proteins.The screened protein was preliminarily verified by western blotting.Methods:1.Preparation of the magnetic bead-Shuangshen Ningxin Capsule active component complexThrough careful screening of references and preliminary experiments,dehydrocorydaline and tetrahydropalmatine were selected as the effective components of Shuangshen Ningxin Capsule to prepare the magnetic bead-Shuangshen Ningxin Capsule active component complex.The magnetic beads and the activated drug monomers were placed in the coupling buffer solution for coupling and binding,and the unbound drug monomers were eluted after full reaction to obtain the magnetic beaddrug complex.Then the magnetic bead-drug complex was judged by HPLC-NMR to determine the binding situation between the magnetic beads and drug monomers,and then could be used for follow-up experiments.2."Fishing" and separation of the target protein of the active component of Shuangshen Ningxin CapsuleThe magnetic bead-drug complexes of dehydrocorydaline and tetrahydropalmatine were mixed with the protein solution of SD rat heart tissue,mouse myocardial microvascular endothelial cell and rat H9c2 myocardial cell,respectively.The target proteins were "fishing".After "fishing",the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.3.Identification and screening of target proteins of effective components of Shuangshen Ningxin CapsuleThe proteins were separated from the gel by protease hydrolysis,and then the proteins were identified by HPLC-MS to obtain the detailed information.The range of screening proteins was narrowed by bioinformatics analysis and molecular docking technology,to determine the target protein.4.Verification of the target protein of the effective component of Shuangshen Ningxin CapsuleWestern blotting was used to verify the direct target of effective components of Shuangshen Ningxin Capsule in preventing myocardial ischemia reperfusion injury.Results:1.Preparation of the magnetic bead-Shuangshen Ningxin Capsule active component complexMagnetic bead-dehydrocorydaline complex and magnetic beadtetrahydropalmatine complex were prepared successfully.The binding rates of magnetic beads to drug monomers were 91.79%and 92.15%,respectively.The binding rates of magnetic beads to drug monomers were good.2."Fishing" and separation of the target protein of the active component of Shuangshen Ningxin CapsuleThe magnetic bead-drug complex was mixed with three protein solutions,"fishing" protein and the protein was well isolated by SDS-PAGE.The specific protein was clearly distinguished by Coomasil bright blue staining.For example,the molecular weights of the proteins binding to the histones of SD rat heart tissue model by dehydrocorydaline magnetic beads and tetrahydropalmatine magnetic beads are about 63kDa,50kDa,35kDa,25kDa.The molecular weights of the proteins binding to the histones of MMVECs model were about 87kDa,53kDa,45kDa,32kDa,25-28kDa,21kDa.The molecular weights of the proteins binding to the histones of H9c2 model were about 100kDa,64kDa,53-58kDa,45kDa,30-33kDa.and 15kDa.3.Identification and screening of target proteins of effective components of Shuangshen Ningxin CapsuleDehydrocorydaline and tetrahydropalmatine were bound to three different proteins,respectively.Mass spectrum identification showed that dehydrocorydaline was bound to 1135 SD rat heart histones,1060 MMVECs cell model histones and 239 H9c2 myocardial cell model histones.Tetrahydropalmatine was bound to 926 SD rat heart histones,2059 MMVECs cell model histones,and 438 H9c2 cardiomyocyte model histones.After preliminary screening,enrichment analysis found that most proteins were involved in glucose,fatty acid,protein,energy and other metabolic pathways.Venn analysis and molecular docking finally screened Etfa,Glud1,Acadm,Atp5f1a,P4hb,Vim and other proteins.4.Verification of the target protein of the effective component of Shuangshen Ningxin CapsuleEtfa was considered to be a direct target for the effective components of Shuangshen Ningxin Capsule to prevent myocardial ischemia reperfusion injury by western blotting.Conclusion:1.Magnetic bead-dehydrocorydaline complex and magnetic bead-tetrahydropalmatine complex were prepared successfully.2.The binding proteins of effective components of Shuangshen Ningxin Capsule,dehydrocorydine and tetrahydropalmatine,mostly related to the metabolic pathways such as glucose,fatty acid,protein and energy,and Etfa,Glud 1,Acadm,Atp5f1a,P4hb and Vim might be the main target proteins.3.Etfa may be a direct target for the effective components of Shuangshen Ningxin Capsule to prevent myocardial ischemia-reperfusion injury. |
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