Study On The Anti-platelet Aggregation Mechanisms Of Active Monomers From Corydalis By Differential Proteomic Analysis

Platelet hyperactivation is one of the significant causes of thrombosis,while most cardiovascular and cerebrovascular diseases such as atherosclerosis and coronary heart disease are closely related to thrombosis.Therefore,the development of anti-platelet drugs is one of the important means for the prevention and treatment of cardiovascular and cerebrovascular diseases.Rhizoma Corydalis(RC),a traditional Chinese medicine for promoting blood circulation and removing blood stasis.Dehydrocorydaline,canadine,tetrahydrocoptisine,corydalineandglaucine are the main anti-platelet aggregation active ingredients in RC,nevertheless,the specific mechanism of action is still unclear.Since platelets do not contain a nucleus,retain low levels of mRNA to translate,thereby making the proteome analysis of platelets especially important,and analysis of the changes in the proteome induced by drug is an ideal way to study the drug actions.This thesis applies the platelet differential proteomestudy based on the established two-dimensional electrophoresis(2-DE)system combined with mass spectrum(MS)to explore the antiplatelet aggregation mechanism of active components in RC.The thesis includes four main chapters.The first part is literature review.In this part,the applications of proteomics in the study of cell signaling pathways in tumors,liver diseases,metabolism and pathogenesis of pathogenic microbes were summarized.Then the research progress of RC and its active components on anti-platelet aggregation were presented.The second part is the differential proteome study of anti-platelet aggregation mechanisms of active monomers from RC.Firstly,the in vitro inhibition effect of the four active compounds including dehydrocorydaline,canadine,tetrahydrocoptisine and corydaline on different agonists including thrombin(THR),adenosine diphosphate(ADP)and arachidonic acid(AA)induced platelet aggregation were tested by turbidimetry method.And the results revealed that dehydrocorydaline and canadine could dosedependently inhibit platelet aggregation,while tetrahydrocoptisine and corydaline had a fairly weak effects.Furthermore,dehydrocorydaline and canadine had the strongest effect on ADP-and THR-induced platelet aggregation,respectively.Secondly,the differential proteome analysis was performed.The platelet proteins after treated with the monomers or blank solvent were separated by 2-DE.Subsequently,MALDI-TOF-MS/MS was used to identify the differentially expressed proteins between the drug-treated and corresponding control group.The results showed that the treatment of active compounds changed the expressions of about 110 proteins in platelet(the fold changed are more than 1.8),which provides relevant information for the further prediction of the drug action mechanisms.In addition,diacylglycerol(DAG)is an important chemical information molecule invoved in Gq pathway,and exercises the function of carrying and amplifying signals in platelet activation.Thus enzyme linked immunosorbent assay(ELISA)test was used to determine the effect of drug on DAG expression and the results showed that dehydrocoryline could reduce the content of DAG in platelets after the platelet activation.The third part is to explore the anti-platelet mechanism of the active components from RC.The functional analysis of the identified differentially expressed proteins were performed through literatural searching and the results showed that they are mainly involved in cytoskeletal,energy metabolism,cell signaling and so on.The proteins invoved in signal pathways of platelet activation are closely related to the mechanism of drug action.Along with the known specific signal transduction pathways of platelet activation,it was speculated that the dehydrocorydaline might inhibit platelet aggregation by binding with the ADP receptors P2Y1 and P2Y12,and the most likely signal is through activation of RhoA/MLC2 pathway to make reorganization of the platelet cytoskeleton.The effect of canadine on platelet function may be related to its binding to THR receptor PAR1 for mediated Gi signaling pathway.However,the tetrahydrocoptisine and corydaline require further studies to confirm their actions.The fourth part is the conclusion and prospective.In this paper,the possible antiplatelet aggregation signaling pathway of two active monomers dehydrocorydine and canadine in the RC were predicted by differential proteomicanalysis,providing a basis for further study on the target proteinsof the monomers and the anti-thrombotic mechanism of RC.