Research On Serum Proteomics For Hot-dampness And Blood Stasis Symdrome Of Rheumatoid Arthritis Based On ITRAQ And Magnetic Beads Enrichment

BackgroundRheumatoid arthritis(RA)is an common autoimmune disease,which usually affect the joints.It is one of the important causes of disability.In Traditional Chinese Medicine RA belongs to the "Bi condition","Lijie"and "aggravated bi".It is effective,cheap,individualized and safe to treat RA with Chinese medicine.Hot-dampness and blood stasis is the important syndrome type of RA,especially for active RA.Clearing heat and activating blood circulation is the main therapeutic method for Hot-dampness and blood stasis of RA.Accurate syndrome differentiation guarantees the clinical curative effect.So it is a big problem of Diversification and subjectivization of syndrome differentiation which influence the effect of drug application.It is a major aspect of objective research of TCM syndrome for the combination of Chinese traditional and Western medicine.To some extent TCM syndrome is the response of pathological state.On account of the integrality,Ambiguity,variability,diversity and complexity of TCM syndrome,single physical and chemical indicators can not embody the meaning of TCM syndrome.So it is a difficult problem to objectify TCM syndrome for the combination of Chinese traditional and Western medicine.The development of systems biology,typically proteomics,bring systematic thinking to modern medicine on the basis of reductionism.Systematic thinkingmake it possible to objectify TCM syndrome.It coincides with TCM in the concept of integrality,variability,informatization and complexity.Proteomic technology has been be widely used in the reaserch of TCM and made a lot of achievement.Now there is no research on hot-dampness and blood stasis of RA using proteomic technology.Our research intend to explore on the physical basis of hot-dampness and blood stasis of using iTRAQ,UPLC-QE-MS,magnetic beads enrichment and MALDI-TOF/TOF-MS.Then it can guide the clinical diagnosis and treatment.RESEARCH ONE The proteomic Analysis and verification of hot-dampness and blood stasis of RA based on iTRAQ.Purpose:Search for differential proteins of hot-dampness and blood stasis syndrome of RA,research the pathogenesis and guide the clinical diagnosis and treatment.Methods:Our research includes 90 cases,divided into hot-dampness and.blood stasis syndrome,non-hot-dampness and blood stasis syndrome and healthy volunteers.Each group includes 30 cases,which has 4 men and 26 women.By H test with SPSS v 19.0 software,there is no difference among three groups.Serum samples are mixed in each group,then tested by iTRAQ and UPLC-QE-MS.The mass spectrometric data is analyzed by Thermo Proteome Discoverer 2.1 software.The detected protein is supposed to be differential protein when fold change>1.5 or ≤0.7 and q-value<0.05.DAVID Bioinformatics Resources is used to analysis Gene ontology annotation,Function of clustering analysis and KEGG Pathway.STRING is used to analysis protein-protein interaction.Results:Compared with healthy volunteers,there are 72 differential proteins acquired in hot-dampness and blood stasis syndrome,among which 25 proteins are up-regulated and 47 proteins are down-regulated.While there are 12 differential proteins acquired in non-hot-dampness and blood stasis syndrome,among which only 1 proteins are up-regulated and 11 proteins are down-regulated.There are obviously differences between hot-dampness and blood stasis syndrome and non-hot-dampness and blood stasis syndrome.According to Gene ontology annotation of hot-dampness and blood stasis syndrome,the Molecular functions of the up-regulated proteins include 9 aspects,such as acute-phase response,Fc-gamma receptor signaling pathway involved in phagocytosis,receptor-mediated endocytosis,complement activation,platelet degranulation,complement activation,classical pathway,platelet aggregation,proteolysis,blood coagulation,fibrin clot formation.The cellular component has 10 aspects,of which extracellular region,extracellular space,blood microparticle and extracellular exosome account for 78%.2 biological processes are acquired,antigen binding and serine-type endopeptidase activity.The Molecular functions of the up-regulated proteins include 26 aspects,such as complement activation,classical pathway,complement activation,receptor-mediated endocytosis,regulation of immune response,Fc-gamma receptor signaling pathway involved in phagocytosis,proteolysis and so on.The cellular component has 12 aspects,including extracellular region,blood microparticle,extracellular exosome,extracellular space and so on.10 biological processes are acquired,such as serine-type endopeptidase activity,antigen binding,cholesterol binding,high-density lipoprotein particle receptor binding.There are 6 proteins related to acute-phase response in up-regulated proteins of hot-dampness and blood stasis syndrome,including Serum amyloid Al,a 1-acidglycoprotein,C-reactive protein,Haptoglobin,Serum amyloid A4 and a 2-acid glycoprotein.4 Immunoglobulin fragments,IGHG3、IGHV1-69、IGHV4-34、IGHL7-43 are related with complement activation.Fibrinogen alpha chain,Actin β,and Fibrinogen gamma chain participate in platelet degranulation,platelet aggregation,blood coagulation,fibrin clot formation.In addition,Fibrinogen gamma chain,Fibrinogen alpha chain,a 1-acid glycoprotein and a 2-acidglycoprotein are related to platelet degranulation.All these mean that hot-dampness and blood stasis syndrome is closely associated with acute inflammation and Coagulation disorders.Signal pathways such as Coagulation and complement cascades,Bacterial invasion of epithelial cells,Shigellosis,Platelet activation are found to activate by KEGG Pathway analysis in up-regulated proteins of hot-dampness and blood stasis.There are 17 proteins of hot-dampness and blood stasisthat directly participate in Pathogenesis of RA identified by available literature.Conclusions:By comparing RA hot-dampness and blood stasis syndrome,RA non-hot-dampness and blood stasis syndrome and healthy volunteers,a series of differential proteins were screened.In hot-dampness and blood stasis syndrome differential proteins,acute inflammatory proteins and blood coagulating proteins take a large part.It indicates an important relationship between RA hot-dampness and blood stasis syndrome and disease activity.It is similar to some research about hot-dampness syndrome.It provides evidences for the using of heat-cleaning and blood-activating therapy in RA.It is significant to research the material basis of hot-dampness.and blood stasis of RA by iTRAQ technology.According to these differential proteins,we can get more views to explore the mechanism of RA and hot-dampness and blood stasis syndrome.And we can make our drug use more precise.RESEARCH TWO The establishing and verification of disaggregated model for RA and hot-dampness and blood stasis syndrome serum peptidome pattern based on magnetic beads enrichment.Purpose:Establish the model of RA and hot-dampness and blood stasis serum peptidome pattern,to guide the clinical therapy.Methods:Our research includes 40 RA cases,divided into hot-dampness and blood stasis syndrome and non-hot-dampness and blood stasis syndrome.Each group includes 20 cases,which has 2 men and 18 women.20 healthy volunteers are enrolled as normal group.By H test with SPSS v 19.0 software,there is no difference among three groups.We make serum peptidome pattern model of RA with 40 RA cases and 20 healthy volunteers,while make model of hot-dampness and blood stasis syndrome of RA with 20 hot-dampness and blood stasis syndrome cases and 20 non-hot-dampness and blood stasis syndrome cases.Magnetic beads enrichment technology is used to separate serum sample peptide.After targeting and drying,the serum sample peptides are detected by MALDI-TOF/TOF-MS.The masss pectrometric data is analyzed to make disease classification modelby Clinprotools 3.0 software.Other 30 cases,including 10 hot-dampness and blood stasis syndrome of RA,10 non-hot-dampness and blood stasis syndrome of RA and 10 healthy volunteers are enrolled to verify the accuracy of RA model.20 RA cases and 10 healthy volunteers are used to verify the accuracy of RA serum peptidome pattern model,while 10 RA hot-dampness and blood stasis syndrome and 10 RA non-hot-dampness and blood stasis syndrome are used to verify the accuracy of serum peptidome pattern modelfor hot-dampnessand blood stasis syndrome.The processing method of serum sample is as same as above.The masss pectrometric data is loaded into CPT software to calculate the accuracy rate.Results:The model of RA serum peptidome pattern by GA-5 algorithm is considered to be the optimal,composed of 5 peptide with m/z 2601.05,4967.6,6639.43,1866.29,8144.63.There are statistical differences between groups with m/z 4967.6,6639.43,8144.63(P<0.000001).The expression level of peptides with m/z 4967.6,6639.43 is positively correlated with RA,while the expression level of peptides with m/z 8144.63 has a negative correlation with RA.The recognition rate of this model is 94.74%,and its cross validation rate is 81.96%.After verification it has a accuracy rate of 97.5%.The peptide with m/z 4967.6,6639.43 has the value of biomarker to research the mechanism of RA.The RA hot-dampness and blood stasis syndrome model by GA-7 algorithm is considered to be the optimal,composed of 5 peptide with m/z 5:740.43,4251.35,2863.03,1607.22,4531.5.There are statistical differences between groups with m/z 5740.43(P<0.000001).Its expression level is positively correlated with hot-dampness and blood stasis syndrome of RA.The recognition rate of this model is 97.50%,its cross validation rate is 80.05%.After verification it has a accuracy rate of 90%.The peptide with m/z 5740.43 has the value of biomarker to research the mechanism of RA hot-dampness and blood stasis syndrome.Conclusions:The serum peptidome pattern models established show a high precision to figure out RA and RA hot-dampness and blood stasis syndrome.Our research lay a foundation for the further study of the disease mechanism.