Blood stasis syndrome:clinical multi-omics research and related problems

Min Li;Rui-Jin Qiu;Hong-Cai Shang＊

1 Department of Cardiology,Beijing University of Chinese Medicine Third Affiliated Hospital,Beijing,100029,China.

2 Key laboratory of Chinese Internal Medicine of Ministry of Education and Beijing,Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing,China.

Abstract Multi-omics is a biological analysis approach in which the data sets are multiple"omics",such as genomics,transcriptomics,proteomics,metabolomics.Multiomics can be used in the detection,diagnosis,treatment,and prognosis of diseases as a kind of potential biomarker.Blood stasis syndrome(BSS)is a common syndrome in traditional Chinese medicine(TCM).Multi-omics can reflect the change of BSS as a whole.Thus,the purpose of this manuscript was to fully review the clinical multi-omics research and related problems of BSS.We found that the multi-omics of BSS reflected many pathophysiological processes through regulating vascular endothelial cells,vasodilation and contraction,oxidative stress,thrombosis,angiogenesis,platelet aggregation,lipid metabolism,inflammatory response,immune and coagulation function,energy balance,amino acid metabolism,and so forth.Many potential biomarkers of BSS were found in multi-omics and some promoting blood circulation Chinese medicines(PBCCMs)could well regulate the abnormal biomarkers and make them return to the normal level.

Keywords Blood stasis syndrome,Multi-omics,Genomics,Transcriptomics,Proteomics,Metabolomics

Background

Syndrome differentiation and holistic view are the main features of traditional Chinese medicine(TCM).“Syndrome” is a special thinking method of cognition and treatment of diseases in TCM,which can summarize changes in a certain stage,reflecting TCM's understanding of disease[1].Blood stasis syndrome(BSS)is a major classification of "syndrome".In TCM theory,BSS means that the blood is stagnated at a certain place and cannot be dissipated in time,resulting in the lump,pain,bleeding,and so on[2].The study of blood stasis has a long history.There are related descriptions of BSS inHuang Di Nei Jing,and in the Eastern Han Dynasty,Zhang Zhongjingfirst put forward the term "blood stasis" in his book(Jin Gui Yao Lue).After the continuous enrichment and development of later doctors,the theory of BSS had been improved in the Qing Dynasty.Wang Qingrenhas made a great contribution to developing BSS and has created more than 30 related prescriptions,which are widely used in clinical practice.In the past half-century,the number of BSS research has been increasing,attracting more and more attention from scholars of traditional Chinese and western medicine.Many promoting blood circulation Chinese medicines(PBCCMs)have also been widely used,such as Xuefu Zhuyu Decoction.Multi-omics is a biological analysis approach in which the data sets are multiple"omics",such as genomics,transcriptomics,proteomics,metabolomics.Multi-omics can be used in the detection,diagnosis,treatment,and prognosis of diseases as a potential biomarker.In modern research,multi-omics can reflect the change of BSS as a whole and is related to BSS’s pathophysiology[3].

BSS and genomics

Genomics

The gene is the basic physical and functional unit of heredity.Genes are made up of DNA.Some genes act as instructions to make molecules called proteins.However,many genes do not code for proteins.A genome is an organism's complete set of DNAs,including all of its genes.In humans,the genome includes both the genes(the coding regions)and the noncoding DNA and mitochondrial DNA.The study of the genome is called genomics.The term genomics was coined by Thomas Roderick(1986),but genomics was established by Frederick Sanger when he first sequenced the complete genome of a virus and a mitochondrion[4].Genomics includes structural genomics,functional genomics,and comparative genomics.Structural genomics reflects the physical nature of genomes and includes the sequencing and mapping of genomes.Functional genomics mainly studies the expression and function of the genome,focusing on dynamic aspects such as gene transcription,translation,regulation of gene expression,and protein-protein interactions.Comparative genomics involves comparing the genomes of different organisms and it can be used to define important structural sequences that are identical in many genomes and to detect evolutionary changes across genomes.Genomics focuses on interactions between loci and alleles within the genome and other interactions such as epistasis,pleiotropy,and heterosis.Genomics also involves the sequencing and analysis of genomes through the uses of high throughput DNA sequencing and bioinformatics to assemble and analyze the function and structure of entire genomes[5-6].

Application of gene polymorphism and epigenomics in BSS of TCM

Gene polymorphism refers to the existence of two or more discrete alleles in a biological population,also known as genetic polymorphism.A gene is considered to be polymorphic if it occupies more than one allele in a population.Gene polymorphisms can occur in any region of the genome.The majority of polymorphisms are silent,which means that they do not alter the function or expression of the gene.Polymorphism at the DNA level includes a wide range of variations from single base pair change,many base pairs,and repeated sequences.Gene polymorphism can be present in many forms,including a variable number of tandem repeats,transposable elements,structural alterations,copy number variations,and single nucleotide polymorphisms(SNPs).SNPs are single nucleotide changes that happen in the genome in a particular location.The single nucleotide polymorphism is the most common form of genetic variation[7].

Coronary heart disease(CHD),also known as coronary artery disease(CAD)or ischemic heart disease(IHD),involves the reduction of blood flow to the heart muscle due to the build-up of plaque in the arteries of the heart.BSS plays an important role in the formation and development of CHD.CHD-BSS is the most common syndrome of cardiovascular diseases.There was no significant correlation between CHD-BSS and angiotensin-converting enzyme(ACE)/endothelial nitric oxide synthase(eNOS)gene polymorphism,but the severity of patients with DD and I/D type of ACE gene is higher than type II,and there were multiple lesions[8,9].Compared with the non-BSS and healthy control group,the frequency of ACE DD genotype and D allele,endothelin(ET),and angiotensin Ⅱ(Ag Ⅱ)were higher in the BSS group,and the DD type of ACE gene might be the susceptible gene of CHD[10].The rs4762 and rs699 polymorphism of angiotensinogen(AGT)were correlated with the CHD-BSS in Hunan Province.The rs4762 polymorphism and the rs699 polymorphism CC-type might be the susceptible genes of the CHD-BSS in Hunan Province[11].Myocyte Enhancer Factor 2A(MEF2A)polymorphism loci(rs2033547 and rs34851361)were not the susceptibility loci of CHD-BSS[12].Xuefu Zhuyu oral liquid could reduce blood stasis symptoms,improve blood rheology in patients with CHD-BSS,this clinical effect is related to the polymorphism of platelet membrane glycoprotein Ⅱ b(GPⅡ b),human platelet antigen 3(HPA-3)[13].Compared with the non-BSS group,CYP2C19*2 gene mutation was significantly higher in the BSS group,CYP2C19*2 gene mutation may produce blood stasis by reducing the effect of clopidogrel[14].The rs1323288 and rs2760501 polymorphisms of the activation protein-1(AP-1)were not associated with genetic susceptibility to ischemic stroke[15].Methyl tetrahydrofolate reductase(MTHFR)677 CT/TT genotype and high levels of blood homocysteine(Hcy)were risk factors for deep venous thrombosis(DVT)in Fujian,which may be one of the susceptible factors for the formation of DVT-BSS[16].The ATP-binding cassette,sub-family A,member 1(ABCAl)219KK genotype and G protein subunit beta 3(GNB3)825TT genotype in CHDBSS patients were significantly different from those in non-BSS patients and healthy participants[17,18].

Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell,known as the epigenome.Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence.Two of the most characterized epigenetic modifications are DNA methylation and histone modification[19].Compared with the healthy group,the promoter methylation status of the Kruppel like factor 5(KLF5)and LDL receptor-related protein 12(LRP12)was not significantly different in patients with CHD-BSS[20].In the sequence of 2000bp before the transcriptional start site of the gene Interleukin-6(IL-6),BSS samples were exhibited higher than in the non-BSS group at a total of 7Cpg sites from -1118bp to -826bp,whereas no significant difference in methylation level between the two groups was observed in the region(from -1471bp to -1184bp)[21].There were 128 differential hydroxymethylation genes and 30 pathways related to BSS in CHD.All the genes and pathways are mainly related to the energy metabolism of myocardial cells,inflammation,plaque formation,endothelial injury,immune response,and lipid metabolism.The degree of hydroxymethylation of ADP-ribosylation factor 6(ARF6)and pleckstrin-sec7 domain 2(PSD2)genes was lower in CHD with BSS,which may be the lower hydroxymethylation level of PSD2 gene through PSD2-GEFs-ARF6-small GTPase ARF6 signal transduction pathway regulates the expression of low hydroxymethylation of ARF6 gene.Therefore,ARF6 and PSD2genes participate in the regulation of risk factors such as lipid accumulation,vascular endothelial injury with BSS in CHD[22].

Gene polymorphism and epigenomics were the main aspects of genomics for BSS.The corresponding clinical trials were focused on CHD,stroke,DVT,and so forth.The genes were related to regulating vascular endothelial function,oxidative stress,vasodilation and contraction,thrombosis,platelet aggregation,lipid metabolism,inflammation,and so on.Most epigenomic studies on methylation had no statistical significance,while genotype and polymorphism studies exhibited the genomic differences between BSS and non-BSS.

BSS and transcriptomics

Transcriptomics

The transcriptome is the set of all RNA molecules in one cell or a population of cells.The information content of an organism is recorded in the DNA of its genome and expressed through transcription.Transcriptome broadly refers to the set of transcriptional products of all genetic information in an organism or a specific tissue or cell,that is,the sum of all RNAs,including messenger RNA(mRNA)and non-coding RNA(ncRNA).In the narrow sense,it means the set of all mRNAs.In recent years,ncRNAs have received extensive attention,including small nuclear molecule RNA(snRNA),nucleolus small molecule RNA(snoRNA),microRNA(miRNA),long non-coding RNA(1ncRNA),and circular RNA(circRNA),which play an important regulatory role in the occurrence and development of a variety of diseases.Transcriptomics is the study of the transcriptome-the complete set of RNA transcripts that are produced by the genome under specific circumstances or in a specific cell-using high-throughput method,such as microarray analysis[23-25].

Application of transcriptomics in BSS of TCM

Gene expression profilingmeasures mRNA levels,showing the pattern of genes expressed by a cell at the transcription level.This often means measuring relative mRNA amounts in two or more experimental conditions,then assessing which conditions resulted in specific genes being expressed.Different techniques are used to determine gene expression.These include DNA microarrays and sequencing technologies.The former measures the activity of specific genes of interest and the latter enables researchers to determine all active genes in a cell[26].By comparing the genes of CHD-BSS patients,non-CHD-BSS patients,and healthy volunteers,26 upregulated genes and 22 down-regulated genes were screened out.Differential genes are mainly related to inflammatory immune responses[27].In addition,Baolin Yang found that differential genes between CHD-BSS and non-CHD-BSS regulated the pathophysiology of lipid metabolism,blood cohesion,apoptosis,endothelial damage,and arteriosclerosis[28].Hypertension,also known as high blood pressure(HBP),is a long-term medical condition in which the blood pressure in the arteries is persistently elevated.In most people with established essential hypertension,increased resistance to blood flow accounts for the high pressure while cardiac output remains normal.Blood stasis is one of the common pathogeneses of hypertension.In addition,platelet aggregation and thrombosis are closely related to hypertension and blood stasis[29-30]。 Differential genes related to hypertension-BSS were related to stress response,cell differentiation,apoptosis,autophagy of vascular endothelial cells,and immunity[31].

MicroRNAMicroRNA is a small non-coding RNA molecule(containing about 22 nucleotides)found in plants,animals,and some viruses,which plays key roles in the pathogenesis of the human disease.The first miRNA gene,lin-4,was discovered in Caenorhabditis elegans by Victor Ambros and Gary Ruvkun in 1993[32-33].However,microRNAs were not recognized as a distinct class of biological regulators until the early 2000s[34].MicroRNAs act as post-transcriptional regulators of their mRNA targets.Recognition of target mRNAs by partial sequence complementarity to the miRNA results in posttranscriptional gene repression by some combination of transcript degradation and translational inhibition[35-36].The relative expression of miR-208a-3p was upregulated in the acute coronary syndrome(ACS)-BSS group compared with the healthy control group[37].Besides,specific microRNAs associated with CHD-BSS were miR-320c and miR-17-5P[38].

MicroRNA-mRNAMicroRNAs generally bind to the 3'-UTR(untranslated region)of their target mRNAs and repress protein production by destabilizing the mRNA and translational silencing.BSS has been considered to be the major type of syndromes in unstable angina(UA)patients.Jie Wang and Gui Yu identified 1081 mRNAs and 25 miRNAs differentially expressed between UA-BSS patients and healthy participants through microarrays.They found that up-regulation of miR-146b-5p may cause down-regulation of CALR to attenuate inflammation,while up-regulation of miR-199a-5p may induce the downregulation of TP53 to inhibit apoptosis in UA-BSS patients.The expression patterns of miR-146b-5p,miR-199a-5p,CALR,and TP53 were confirmed by qRT-PCR in an independent validation cohort including UA-BSS patients,non-UA-BBS patients,and healthy controls.miR-146b-5p,miR-199a-5p,CALR,and TP53 may be important biomarkers of UA-BSS patients[39].There are DNA methylation sites,differential gene expression profiles of microRNA and mRNA in CHD-BSS.The function of related genes is enriched in immune and inflammatory response pathways.The differential genes have mutual regulatory relationships,which can construct a DNA methylation-microRNA-mRNA regulatory network related to CHD-BSS,and extract the key pathway miR194 promoter-miR194-MAPK signaling pathway.Through randomized controlled trials,it is further confirmed that the key nodes of the miR194 promoter-miR194-MAPK signaling pathway will change after the improvement of coronary heart disease blood stasis syndrome.The key nodes of the miR194 promoter-miR194-MAPK signal pathway would change by the treatment of Xuesetong which can activate blood circulation and remove blood stasis[40].

lncRNA-microRNA-mRNAlncRNA is a type of RNA that is defined as transcripts longer than 200 nucleotides.They are not translated into proteins and have been reported to participate in many cellular processes,such as transcription and post-transcriptional regulation.A large number of studies have shown that lncRNAs can be combined with microRNAs as competitive endogenous RNAs(ceRNAs),thereby affecting and regulating the expression of mRNAs and target genes[41].Through high-throughput sequencing technology and bioinformatics analysis,it was found that the differentially expressed genes of CHD-BSS were mainly related to immunity and inflammation.In the lncRNA-microRNAmRNA regulatory network related to CHD-BSS,three lncRNAs(CTA-384D8.35,CTB-114C7.4,RP11-567M16.6)and one microRNA(miR-3158-3p)were considered as key nodes in the network[42].

In the transcriptomics study of BSS,the corresponding clinical trials have mostly focused on cardiovascular diseases,especially CHD,acute coronary syndrome,and hypertension.From the single aspect of microRNA or mRNA to the linear extension of microRNA-mRNA and lncRNA-microRNA-mRNA,and then through KEGG and GO analysis,the transcription study realizes the network expansion of nodes and the in-depth exploration of signal transduction pathways.According to the above analysis,BSS was mainly related to inflammation,immune response,apoptosis,and so forth.Inflammatory and transcription factors result in microcirculation disorders and hemorheology changes.Chemokines,interleukincytokines,natural killer factors,and complement molecules are involved in tissue cell injury and vascular destruction,leading to the metabolic changes of ischemia and hypoxia.Through transcriptome studies,some lncRNA,microRNA,and mRNA may be potential biomarkers for BSS,while some proprietary PBCCMs could regulate these abnormal biomarkers[38,43]。

BSS and proteomics

Proteomics

The proteome is defined as the entire set or complement of proteins that are or can be expressed by a cell,tissue,or organism.Marc Wilkins coined the term proteome in 1994 in a symposium held in Siena in Italy,it appeared in print in 1995[44-45].However,unlike the genome,the proteome can be different in different tissues,and its diversity corresponds to the diversity of life phenomena[46].Proteomics is the large-scale study of proteomes.It covers the exploration of proteomes from the overall level of protein composition,structure,and activity.It is an important component of functional genomics.Proteomics mainly includes interaction proteomics,constituent proteomics,and comparative proteomics.The process of proteomics is mainly divided into three steps:separation,identification,and analysis.With the improvement of proteomics techniques,related studies have been greatly promoted[47,48].

Application of proteomics in BSS of TCM

The CHD-BSS mainly involved the disorder of inflammatory reactive protein and lipid metabolism.These proteins,such as retinol-binding protein 4,apolipoprotein E,apolipoprotein A1,CD5 antigenic protein,haptoglobin,and serum albumin,may be closely related to CHD-BSS[49].Compared with healthy controls,patients with CHDBSS had 3 down-regulated proteins and 6 up-regulated proteins.The up-regulated proteins mainly included immunoglobulin,fibrinogen,and granular enzyme,while the down-regulated protein included cell surface glycoproteins[50].There were 13 protein differences in the CHD-BSS group,among which CD41 and Actin Y were the marker proteins[51].Compared with non-CHDBSS patients,the content of coagulant protein and cytoplasmic calcium ion was significantly higher in patients with CHD-BSS.This was highly correlated with the expression of platelet membrane protein CD62p,which further affects the formation and development of CHDBSS[52].The CHD-BSS may be related to inflammatory response,and its pathogenesis was caused by myocardial damage,abnormal coagulation factors,lipid metabolism disorders,and oxygen transport disorders[53].There were 16 differentially expressed proteins in patients with BSS of chronic heart failure(HF),among which 11 proteins,such as apolipoprotein E and galactose-3 binding protein,were up-regulated,while 5 proteins,such as vitamin D binding protein and trypsin inhibitor,were down-regulated[54].

IgA nephropathy(IgAN)is a kidney disease that occurs when an antibody called immunoglobulin A(IgA)builds up in your kidneys.This results in local inflammation that,over time,can hamper your kidneys' ability to filter waste from your blood.Specifically,it is a form of glomerulonephritis or inflammation of the glomeruli of the kidney.By comparing the IgAN-BSS,non-IgAN-BSS,and healthy controls,it was found that 7 proteins were the most appropriate biomarkers for the diagnosis of IgANBSS.The M/Z 8713.48 protein was highly expressed in the IgAN-BSS group,and this protein expression was decreased afterleech(a kind of PBCCMs)treatment.It was speculated that the M/Z 8713.48 protein might be the specific protein of IgAN-BSS[55-56].One study had also shown that the M/Z 2798.06 protein may be a protein marker to distinguish IgAN-BSS and non-IgAN-BSS[57].

In the patients with BSS of ischemic stroke,there were 5 up-regulated proteins(binding globin,SP40/40,vascular Rab-GAP/TBC-containing protein,fibrinogen staple chain,and r-actin)and 1 down-regulated protein(TROVE domain family,member 2)[58].There are 2 differential protein points between the psoriasis Vulgaris and the healthy controls,and patients in different syndromes of psoriasis Vulgaris had different proteins expression[59].In the prothrombic status of recurrent abortion,33 cytokines had significantly different expression in the renal deficiency and BSS group,which involved coagulation,platelet aggregation,inflammation,angiogenesis,and other pathophysiological processes[60].One study showed that Ang-2,Foxp3,and MIF may be related molecular markers of BSS in potential human endometriosis[61].

Proteomics of BSS covered a wide range of diseases,including CHD,HF,ischemic stroke,etc.The proteins were related to immunity,inflammation,platelet aggregation,lipid metabolism,angiogenesis and coagulation,and so forth.The protein biomarkers of BSS varied in different diseases,but the abnormal protein expression returned to normal through the treatment ofleech.

BSS and metabolomics

Metabolomics

The metabolome is the global collection of all low molecular weight metabolites that are produced by cells during metabolism and provides a direct functional readout of cellular activity and physiological status.Metabolomics is defined as the systematic comprehensive analysis of all chemical processes concerning metabolites,providing characteristic chemical fingerprints that specific cellular processes yield.It is an emerging technology that holds promise to inform the practice of precision medicine[62].The steps of metabolomics research include sample collection,pretreatment,data collection,analysis,and interpretation[63].Metabolic samples mainly consist of urine,plasma or serum,saliva,cell and tissue extracts[64].It mainly uses gas chromatography(GC),liquid chromatography(LC),capillary electrophoresis(CE),nuclear magnetic resonance(NMR),mass spectrometry(MS),and other separation and data collection analysis techniques[64-65].

Application of metabolomics in BSS of TCM

Blood SamplesBy comparing the metabolites of CHDBSS,non-CHD-BSS and healthy controls,it was found that cholesterol and xylitol were very likely to be potential metabolic markers of CHD-BSS[66].Different syndromes of CHD had abnormal energy metabolism and amino acid.The content of lactic acid,low-density lipoprotein,n,and very-low-density lipoprotein in the sputum turbid syndrome group was higher,while the content of alanine,high-density lipoprotein,unsaturated fatty acid,and alanine was disorder higher in the BSS group[67].Some results showed that valine and acetone may be specific metabolites of CHD-BSS,but further studies with more samples will be needed[68].Four endogenous metabolic markers of CHD-BSS were found,namely citric acid,carnitine,histidine,and xanthine[69].In patients with CHD-BSS,the contents of glucose,taurine,lactic acid,low-density lipoprotein,very low-density lipoprotein,unsaturated fatty acid,d-glucose,and lipid were higher,while the contents of valine,glutamic acid,choline phosphoric acid,and high-density lipoprotein were lower[70].Compared with the UA-BSS group,the contents of lactate,glucose,galactose,n-acetyl-glycoprotein,and lowdensity lipoprotein were higher in the phlegm turbid syndrome group,while the contents of citric acid,highdensity lipoprotein,unsaturated fatty acid,and trimethylamine oxide were lower[71].The metabolism levels of hemolytic phosphatidylcholine,linoleic acid,arachidonic acid,and sphingosine were all down-regulated,indicating that patients with qi-deficiency and BSS of myocardial infarction(MI)had disordered energy metabolism,phospholipid metabolism,and fatty acid metabolism[72].Different syndromes of HF patients have different metabolites.Compared with patients with qi deficiency and BSS,the plasma contents of three lysophosphatidylcholine substances decreased,three carnitine substances increased,two fatty acids grew and creatinine rose in patients with yang-deficiency and waterstagnation[73].

In type 2 diabetic patients with BSS,the levels of acetone,acetoacetic acid,di-hydroxybutyric acid,dihydroxyisopentric acid were higher,while fumaric acid,citric acid,and n-acetylornithine levels were lower.It was concluded that there may be a more serious fat metabolism,amino acid metabolism,and energy metabolism in type 2 diabetic patients with BSS[74].

There were 58 metabolites in the patients with cerebral infarction and BSS,which were mainly amino acids,organic acids,fatty acids,and carbohydrate derivatives,indicating that there was abnormal metabolism of glucose,protein,and lipids[75].In patients with CHD and cirrhosis，seven endogenous metabolites(succinic acid,citric acid,3-hydroxybutyric acid,n-acetyl glycoprotein,trimethylamine oxide glutamine,and histidine)were found to be potential biomarkers of BSS.The abnormal energy and lipid metabolism in patients with BSS could increase oxygen free radicals,promote blood vessel contraction,and cause renal function damage[76].

Urine SamplesThe levels of citric acid,α-ketoglutarate,CIS aconitic acid,3-hydroxybutyric acid,acetone,tyrosine,creatinine,trimethylamine oxide,dimethylamine,and hippuric acid in the phlegm turbid syndrome group were higher than those in the BSS group,while the contents of bile acid and histidine were relatively low[77].The contents of proline,alanine,isoleucine,glucose,valine,histidine,hippuric acid,and other metabolites were increased,while the contents of citric acid,creatinine,and taurine were decreased in patients with CHD-BSS[78].These metabolic changes may be the potential metabolic characteristics of patients with CHD-BSS.Through the metabolomics analysis of urine samples in patients with liver cirrhosis,it found 7 potential metabolic markers,such as alanine,D-glucose,glycine goose deoxycholic acid,lysophosphatidic ethanolamine,citric acid,proline,and bile acid[79].In addition,the metabolic properties of children with different syndromes of cytomegalovirus hepatitis were varied[80].Some evidence showed that dihydroxy prostaglandin,carboxy leukotriene,epigallocatechin,and hydroxybutyric acid may be potential markers of BSS in psoriasis.After the treatment of PBCCM(Yinxieling Optimized Formula),the urine metabolomics was changed obviously[81].

Metabolomics samples of BSS were mainly selected from urine,plasma,or serum,and the related diseases included CHD,MI,HF,type 2 diabetes mellitus,cerebral infarction,and so forth.Abnormal metabolites were related to energy metabolism,prothrombin,phospholipid metabolism,fatty acid metabolism,and amino acid metabolism disorders.

Discussion

Multi-omics studies mainly include genomics,transcriptome,proteomics,and metabolomics,involving cardiovascular diseases(CHD,ACS,MI,HF),respiratory diseases(pneumonia,pulmonary fibrosis),thrombotic diseases(DVT),and so forth.The multi-omics of BSS affected many pathophysiological processes via regulating vascular endothelial cells,inflammatory response,vasodilation,contraction,oxidative stress,thrombosis,angiogenesis,platelet aggregation,lipid metabolism,immune and coagulation function,energy balance,amino acid metabolism,and so on.Finding potential biomarkers for BSS was able to provide evidence for diagnosis and treatment of diseases by multi-omics.Besides,it has been proved that PBCCMs had therapeutic effects to improve multi-omics in the clinics.Based on the above evidence,there were still some problems in multi-omics studies.

There were few studies on the relationship between clinical multi-omics

The central dogma of biology describes the method by which information is taken from genes and used to create proteins.DNA transcription produces RNA,then RNA translation makes proteins.This process is known as gene expression and all life forms use it to create the building blocks of life from genetic information.According to the central dogma,there is an inherent correlation between multi-omics.Current studies have established the connection between the transcriptome(lncRNAmicroRNA-mRNA)and the genome-transcriptome(DNA methylation-microRNA-mRNA),but there were still few studies on the relationship between clinical multi-omics.

The same disease had different biomarkers

Based on the above evidence,we found that there were relatively many studies on BSS of CHD.As for the same disease(CHD),its biomarkers were different,especially in proteomics and metabolomics.There were five possible reasons.Firstly,the individual difference was the main reason.Secondly,the small sample caused bias of results in multi-omics.Thirdly,CHD contains many subtypes,such as stable angina pectoris,unstable angina pectoris,myocardial infarction,and so forth,which could lead to a variety of biomarkers coming into being.Fourthly,different regional humanities would make an impact on the results of multi-omics;Fifthly,different detection methods for multi-omics were also an important point causing multiple biases.Therefore,a large number of samples are needed to verify the multi-omics studies in the future.

Multi-omics studies of PBCCMs for treating BSS was not comprehensive

PBCCMs have good clinical effects on patients with BSS,which enable them to promote blood circulation,dissipate stasis,relieve pain,and regulate menstruation.Based on many multi-omics studies,the BSS contained a large number of potential biomarkers,and some PBCCMs could well regulate the abnormal biomarkers and make them return to the normal level.Currently,there were more studies on multi-omics of BSS,while a few studies have been done on multi-omics of PBCCMs.Therefore,we should conduct more and more multi-omics studies of PBCCMs and further reveal their pharmacological mechanism.

Abbreviations

BSS,Blood stasis syndrome;TCM,Traditional Chinese medicine;PBCCMs,Promoting blood circulation Chinese medicines;SNPs,Single nucleotide polymorphisms;CHD,Coronary heart disease;CAD,Coronary artery disease;IHD,Ischemic heart disease;ACE,Angiotensinconverting enzyme;eNOS,Endothelial nitric oxide synthase;ET,Endothelin;Ag Ⅱ,Angiotensin Ⅱ;AGT,Angiotensinogen;MEF2A,Myocyte Enhancer Factor 2A;GP Ⅱb,Glycoprotein Ⅱb;HPA-3,Human platelet antigen 3;AP-1,Activation protein-1;Hcy,Homocysteine;DVT,Deep venous thrombosis;ABCAl,ATP-binding cassette,sub-family A,member 1;GNB3,G protein subunit beta 3;KLF5,Kruppel like factor 5;LRP12,LDL receptor-related protein 12;IL-6,Interleukin-6;ARF6,ADP-ribosylation factor 6;PSD2,Pleckstrin-sec7 domain 2;mRNA,Messenger RNA;ncRNA,Non-coding RNA;snRNA,Small nuclear molecule RNA;snoRNA,Nucleolus small molecule RNA;miRNA,MicroRNA;1ncRNA,Long non-coding RNA;circRNA,Circular RNA;HBP,High blood pressure;ACS,Acute coronary syndrome;UA,Unstable angina;ceRNAs,Competitive endogenous RNAs;HF,Heart failure;IgAN,IgA nephropathy;IgA,Immunoglobulin A;GC,Gas chromatography;LC,Liquid chromatography;CE,Capillary electrophoresis;NMR,Nuclear magnetic resonance;MS,Mass spectrometry;MI,Myocardial infarction.

Acknowledgments

This work was supported by grants from the National Key Research and Development Program(2019YFC1710403).The project of new teacher’s start-up Fund of Beijing University of Chinese Medicine(2020-JYB-XJSJJ-059).

Author contributions

Hong-Cai Shang provided guidelines for this review.Min Li and Rui-Rin Qiu wrote the main manuscript.

Competing interests

All authors declare that they have no conflicts of interest.