A Survey Of Proteomic Biomarkers For Rats With Qi Deficiency And Blood Stasis Syndrome In Chronic Heart Failure

Qi deficiency and blood stasis syndrome is one of the core theories of TCM syndromes.It is caused by long-term illness and internal stagnation of blood stasis.Heart failure refers to a syndrome that causes structural damage or dysfunction of the heart caused by any reason,resulting in a decrease in the ability of the heart to supply blood and oxygen.Over time,it evolves into chronic heart failure,with qi deficiency and blood stasis.Qi deficiency and blood stasis syndrome is one of the most common syndromes of clinical chronic heart failure.Proteomics based on mass spectrometry technology has promoted the discovery of disease biomarkers at the protein molecular level.Although there are many analytical methods,the discovery of biomarkers is still slow.Therefore,methodological optimization and research are still needed.In addition,there are few proteomics researches on rats with CHF syndrome of Qi deficiency and blood stasis induced by combined coronary artery ligation and intervention of FU FANG REN SHEN BU QI KE LI.In response to these problems,this research first compares and optimizes the proteomic analysis methods of blood samples to provide technical support for proteomics research.Secondly,combined with the foundation of the laboratory’s preliminary work,establish animal models that conform to TCM syndromes and diseases.Preliminary evaluations were carried out with pharmacodynamics,and finally the proteomics characteristics of CHF qi deficiency and blood stasis syndrome were initially explored through proteomics technology based on mass spectrometry,and on this basis,the protein-level regulatory mechanism of FU FANG REN SHEN BU QI KE LI were preliminary studied.Objective:To compare and study the proteomic analysis methods of blood samples,establish and optimize the process suitable for the proteomic analysis of blood samples,and establish a rat animal model of chronic heart failure with Qi-deficiency and blood stasis syndrome based on the preliminary work of the laboratory,complete the model evaluation and FU FANG REN SHEN BU QI KE LI medicine Efficacy evaluation,and optimized blood sample proteomics analysis method to carry out preliminary research on the pathological mechanism of chronic heart failure rats with Qi deficiency and blood stasis syndrome and the therapeutic mechanism of FU FANG REN SHEN BU QI KE LI at the protein level.Methods:1 Comparative study of proteomic analysis methods of blood samplesThe Q-Exactive Plus mass spectrometer was used to compare and analyze the proteome composition of rat blood samples prepared by the pretreatment method of plasma,serum and serum to remove high-abundance proteins;compare the routine enzyme digestion,incubation at 45℃,heat-assisted digestion,and secondary digestion of serum samples.The efficiency of secondary heat-assisted digestion,urea-assisted digestion and variable temperature digestion;compare the qualitative and quantitative characteristics of DDA,DIA and PRM mass spectrometry data collection;use optimized methods for proteomic analysis of rat blood samples.And provide technical support for proteomics research.2 Establishment and evaluation of rat model of chronic heart failure with Qi deficiency and blood stasis syndrome and preliminary exploration of pharmacodynamicsThe left anterior descending coronary artery ligation combined with the water platform chronic sleep deprivation method was used to establish a rat model of chronic heart failure with Qi deficiency and blood stasis syndrome.The treatment was given by intragastric administration of FU FANG REN SHEN BU QI KE LI,and the positive drug was set for intragastric administration of valsartan.Control group.Syndrome indicators(general state,body weight,holding power,tongue R,G,B values,pulse amplitude)and disease indicators(echocardiographic measurement of cardiac function indicators and ventricular remodeling indicators,myocardial tissue pathomorphology measurement)Carry out model evaluation and pharmacodynamic evaluation to lay the foundation for proteomics research.3 Preliminary Study on Serum Proteomics of Rats with Chronic Heart Failure Qi Deficiency and Blood Stasis SyndromeLabel-Free quantitative proteomics technology completes mass spectrometry analysis of rat serum proteome,using optimized method:remove high-abundance proteins,heat-assisted variable temperature digestion,data-dependent acquisition mode to obtain mass spectrometry data,using Proteome Discover and Maxquant software to complete The qualitative and quantitative of RAW files,DEPs and related pathways are enriched by OR value(Odds ratio),Preliminarily reveal the physiological and pathological mechanisms related to disease and syndrome from the protein level.Result:1 Comparative study of proteomic analysis methods of blood samplesAfter removing high abundance protein from serum samples,the number of identified proteins was higher and the quantitative repeatability was better.The number of identified proteins and peptides,and the rate of matched mass spectrum were relatively high when serum samples digested by heat assisted digestion and variable temperature digestion.The efficiency of these two digestion methods and the repeatability of their qualitative and quantitative data were acceptable.Compared the three mass spectrometry methods,DDA is more convenient,DIA is highly reproducible and PRM is more accurate.When removed high abundance protein from serum samples and digested proteins with heat assisted-variable temperature digestion and analyzed by DDA method,490,490and 504 proteins could be identified in three repeated experiments respectively.While a total of 590 proteins were detected,and repetition rate of identified protein was 69.8%.2 Establishment and evaluation of rat model of chronic heart failure with Qi deficiency and blood stasis syndrome and preliminary exploration of pharmacodynamics(1)Syndrome indexesThe rats in the blank control group have smooth and shiny coats,sensitive response and good spirits,and stools are yellowish-brown and striped;the rats in the model group have chlorosis and upright and messy coats,squinting and tiredness and lethargy,slow response,and weak stress response.Compared with the blank control group,rats in the model group have lower body weight(P<0.01),lower holding power value(P>0.05),significant decrease in pulse amplitude(P<0.01),dark red tongue,and significant R and B values Increased(P<0.01 or P<0.05),G value increased(P>0.05);Compared with the model group,the body mass of the drug administration group increased(P>0.05),and the grip value and pulse amplitude increased(P>0.05),the R,G,and B values of the tongue surface decreased(P<0.05),the body weight of the positive drug control group increased(P>0.05),and the grip value and pulse amplitude of rats increased(P<0.05 or P<0.01),the R,G,and B values of the tongue surface decreased(P>0.05).(2)Disease indexesCompared with the blank control group in the same period,the HR of the rats in the normal administration group was reduced,and the SV,EF,FS,and CO of the model group were reduced(P<0.05 or P<0.01);compared with the model group,the SV,EF,FS,CO of the positive drug control group and drug administration group were all increased,and the difference in LVM of each group was not statistically significant.Compared with the blank control group,LVIDs,LVEVs,LVEVd in the model group increased(P<0.05 or P<0.01),LVIDd increased,while LVAWs,LVAWd,LVPWs decreased(P<0.05 or P<0.01),LVPWd decreased;compared with the model group,the LVIDs,LVIDd,LVEVs,and LVEVd of the drug administration group decreased,while the LVAWs and LVAWd increased.The positive drug control group LVIDs,LVIDd,LVEVs,LVEVd,LVAWs,and LVAWd increased.Both LVPWs and LVPWd decreased,and there was no significant difference between the drug administration group and the positive drug control group.The pathomorphological observation of myocardial tissue showed that the myocardial fibers of the blank control group were arranged neatly,orderly,and densely,myocardial cells were not degeneration or necrosis,and there was no inflammatory cell infiltration or fibrous tissue proliferation in the interstitium;myocardial fibers were arranged in the model group.Disordered and disordered,myocardial muscle bundles are widened,myocardial cells are degenerated and swollen,scattered necrosis is seen,interstitial sheet inflammatory cells infiltrate,and fibrous tissue proliferation divides myocardial fibers into islands;the arrangement of myocardial fibers in the drug administration group is loose,Disorder,mild degeneration of myocardial cells,unobvious swelling,occasional necrosis,interstitial vasodilation,congestion,hemorrhage,focal infiltration of inflammatory cells,and reduced fibrous tissue proliferation;positive drug control group:myocardial fibers are arranged loosely and without sequence,myocardial cell swelling is obvious,occasionally necrosis,interstitial vasodilation,congestion,bleeding,focal infiltration of inflammatory cells,and mild hyperplasia of fibrous tissue.After taking the samples,it was observed that in the model group,white watery necrosis at the heart ligation site was visible to the naked eye,and the infarct area was sunken,and some new granulation tissue was added.3 Preliminary Study on Serum Proteomics of Rats with Chronic Heart Failure Qi Deficiency and Blood Stasis SyndromeThrough non-labeled quantitative proteomics,quantitative data of 526 proteins with high credibility in 15 biological samples from 3 biological replicates in 5 groups were obtained.The difference in protein expression was set to be greater than two times to screen for differentially expressed proteins(DEPs)between samples.A total of 675 DEPs were found,which were divided into 422 up-regulated proteins and 253 down-regulated proteins,of which 56 proteins were regulated by FU FANG REN SHEN BU QI KE LI.Syndrome-related pathways,43 proteins related to the model,20 proteins related to FU FANG REN SHEN BU QI KE LI,45 proteins are more likely to respond to FU FANG REN SHEN BU QI KE LI in the model state,57 proteins are regulated by drugs and modeling,63 proteins are thought to be involved in the protein pathways that positive drugs regulate disease,47 proteins were co-regulated in the drug administration group and the positive drug control group.Enrich the pathways of DEPs through GO(Geno Ontology),228 DEPs are enriched in 41 biological pathways;Model-related biological pathways include vesicle-mediated transport,protein interaction,autophagy,signal transduction,stress response,nervous system development,platelet activation and coagulation cascade,cell cycle and mitosis,RNA metabolism,and signaling transduction etc.The proteins expression change after modeling and callback after FU FANG REN SHEN BU QI KE LI treatment are concentrated in the immune system;In the model situation,the proteins with good response value to FU FANG REN SHEN BU QI KE LI mainly involve the assembly,remodeling and clearance of plasma lipoproteins,etc;The expression changes after modeling,and callback after treatment with positive drugs are concentrated on Rap1 signal and integrin signal transduction,activation of MAPK pathway,etc;The proteins co-regulated by FU FANG REN SHEN BU QI KE LI and positive drugs are focused on immune-related pathways.Conclusion1 Comparing the proteomic analysis methods of blood samples,it is found that after removing high-abundance proteins from serum samples,they are digested by heat-assisted combined with variable temperature enzyme digestion,and DDA mass spectrometry analysis technology is used to obtain higher identification protein coverage and better repeatability.Quantitative results,the use of DIA and PRM technology can also be used for subsequent verification of quantitative results.Based on the comparison of blood sample proteomic analysis methods,an optimized preparation and analysis process was established,which created conditions for further proteomics research.2 The general state of rats,body weight,holding power,pulse amplitude,and tongue R,G,B values all indicate that the model group has Qi deficiency and blood stasis syndrome;myocardial tissue pathology,cardiac morphology,and cardiac function The indicators and the ventricular remodeling indicators jointly indicated that the rats in the model group had an obvious state of chronic heart failure.After the intervention of ginseng and astragalus drugs,each indicator had a different degree of callback.3 This experiment analyzed the differentially expressed proteins of the blank control group,normal with drug administration group,model group,model with drug administration group and positive control group through proteomics technology,and designed the normal with drug administration group as a reference to conduct bioinformatics analysis of differentially expressed proteins.Respectively enriched to the protein that may only be related to the rat CHF with qi deficiency and blood stasis syndrome,the protein related to FU FANG REN SHEN BU QI KE LI,the protein that responds to the intervention of FU FANG REN SHEN BU QI KE LI in the case of CHF with qi deficiency and blood stasis syndrome,and FU FANG REN SHEN BU QI KE LI regulates the disease syndrome,the key proteins that positive drugs regulate diseases,and proteins co-regulated by FU FANG REN SHEN BU QI KE LI and positive drugs.CHF with qi deficiency and blood stasis syndrome is enriched in more signal pathways,accompanied by selective autophagy,platelet activation and calcium ion activation,signal transduction and small molecule transport,cell cycle and mitosis,vesicle-mediated transport and cellular stress response,protein metabolism and post-translational modification,gene expression and transcription and other biological processes;in this study,after the intervention of FU FANG REN SHEN BU QI KE LI in CHF with qi deficiency and blood stasis syndrome,most of the disease and syndrome indicators have been adjusted,the results have no significant difference,but they have been significantly enriched.Collected several immune-related pathways and proteins of FU FANG REN SHEN BU QI KE LI regulating CHF qi deficiency and blood stasis syndrome,including Rap1 signaling pathway involved in apoptosis and CD22-mediated BCR regulation,as well as 4 immunoglobulins with Ig domains,YWHAB genes The encoded 14-3-3 protein family and Rap1b,FU FANG REN SHEN BU QI KE LI may regulate CHF qi deficiency and blood stasis syndrome through these proteins and pathways.If the drug intervention time is prolonged,more significant syndromes and disease states and corresponding proteins are expected to appear group mapping to provide support for clinical treatment and Chinese medicine research.