Study On Blood Enrichment Bio-active Components And Mechanism Of Danggui Buxue Decoction Based On Multi-omics

Anemia is an extremely common disease affecting up to one-third of the global population,which shows a condition of low hemoglobin(Hgb).Common measures for the treatment of anemia in clinic include iron,vitamin B12,folate,erythropoietin(EPO),and so on.Nowadays,TCM has attracted more and more attention for the treatment of anemia,in this study,Danggui buxue Decoction(DBD)was mentioned.DBD was first recorded in "Chen Su An Fu Ke Bu Jie"(AD 1127-1131),however,its classic formula was recorded in "Nei Wai Shang Bian Huo Lun"(AD 1247),which nourishing blood effect mainly by invigorating Qi.Therefore,in this study,metabolomics,proteomics,bioinformatics and other approaches were used to explore the internal blood enrichment mechanism and related bioactive components of DBD.Chapter 1.Literature researchThis part systematically reviewed the relationship between spleen,Qi and energy metabolism in TCM,the influence of invigorating Qi herbs and related prescriptions on mitochondria and the original prescription use of DBD.Chapter 2.Integration of network pharmacology approach and molecular docking to investigate the blood enriching mechanism of DBD and predict bioactive componentsSection 1.A network pharmacology approach to investigate the blood enriching mechanism of DBD and predict bioactive componentsIn this study,125 ingredients in Radix Angelicae Sinensis and 87 ingredients in Radix Astragali were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP,http://lsp.nwu.edu.cn/tcmsp.php),after screening with Lipinski's rule of five and ADME process,14 ingredients in Radix Angelicae Sinensis and 23 ingredients in Radix Astragali were finally retained,among which caffeic acid was the common ingredient.517 targets for the therapeutic actions of DBD were obtained from the PharmMapper,Swiss,SEA and STITCH.And then,functions of each target were obtained from UniProt,TTD and published literatures.In addition,protein-protein interaction was also considered.To sum up,49 targets which the active components acted on were identified.These targets were imported to the DAVID Webserver for GO function enrichment analysis and KEGG pathway analysis,Cytoscape was used to visualize the compound-target-pathway network for DBD.47 KEGG pathways which DBD acted on were also come to light.And then,according to KEGG pathway annotation analysis,only 16 pathways seemed to be related to the blood nourishing effect of DBD,such as PI3K-AKT pathway,and so on.Only 32 targets participated in these 16 pathways and they were acted on by 29 of the 36 active compounds.GO analysis results indicated that DBD could promote energy metabolism and mainly regulate inflammatory response,peptidyl-tyrosine phosphorylation and T cell receptor signing pathway via protein binding,CXCR chemokine receptor binding and iron ion binding in cytosol,plasma membrane and intracellular.In this section,ELISA kits were mainly applied to verify the key targets(PTPN6,CXCL2,TGFBR1,IL-2,MET and ITK)predicted in network pharmacology,and PIK3R1,AKT,Pakt,mTOR and pmTOR were chosen to validated the PI3K-AKT pathway by Western Blot The results showed that DBD could significantly improve the peripheral blood indexes and organ indexes in hemorrhagic anemia rats.The pathological results showed that DBD could significantly increase splenic corpuscle volume and lymphocyte count of spleen,and thymic cortex thinning and lymphocyte count of thymus in hemorrhagic anemia rats.ELISA results showed DBD had significant regulatory effects on hematopoietic and immune related targets(PTPN6,CXCL2,TGFBR1,IL-2,MET and ITK)in hemorrhagic anemia rat plasma.The results indicated that the hematopoietic mechanism of DBD might be related to the promoting effects of red blood cells proliferation and differentiation and immune enhancement.However,DBD had no effect on the PI3K/Akt signaling pathwaySection 2.A molecular docking approach to investigate the blood enriching mechanism of DBD and predict bioactive componentsIn network pharmacology studies,the targets of a component,a TCM or a prescription were mianly predicted through several databases,and the main premise and key of the study was to find the accurate components.In this section,the retained 14 ingredients in Radix Angelicae Sinensis and 23 ingredients in Radix Astragali after screening with Lipinski's rule of five and ADME process were simultaneously determined by UPLC-TQ-MS/MS.As senkyunolide K and some other components were not commercial,28 components were finally determined.Finally,a total of 18 components were determined,however,betulinic acid,?-sitosterol,senkyunolide I,coniferyl ferulate,hederagenin,folic acid,kumatakenin and astragaloside I were not determined.Except for the non-commercial components,they included most of the active components predicted in network pharmacology,which indicating that the results of network pharmacology have certain credibility.29 chemical components and 32 targets closely related to anemia in the first section of Chapter 2 were imported into Systemsdock(http://systemsdock.unit.oist.jp/iddp/home/index)for molecular docking,the results showed that docking score? 6.11 contained 14 pairs,docking score>5.52 contained 31 pairs,and docking score? 4.82 contained 35 pairs,however,9 pairs did not receive molecular docking scores.Among them,all targets of astragaloside IV acted on showed effective binding,and all targets of senkyunolide A and senkyunolide K acted on showed good binding.With the results of effective rate,astragaloside IV in Radix Astragali,senkyunolide A and senkyunolide K in Radix Angelicae Sinensis were thought of the major contributing compounds to DBD's blood enriching effect.Combined with determination of bioactive components,effective rate and SystemDock scores,we concluded that astragaloside IV and senkyunolide A were potential active components.Next,docking based on AutoDock was also carried on to clarify the interaction between ligands and receptors.The results showed that the two components were closely binding with the corresponding targets mianly with Hydrogen and van der Waals bondings,among them,the combination of senkyunolide A with MET,and astragaloside IV with ZAP70 were more closely,which suggested that senkyunolide A was more inclined to blood enrichment and immune enhancement,while astragaloside IV was more inclined to immune enhancement,which provided a basis for further pharmacodynamics and molecular mechanism study of the two components.Chapter 3.Integration of organ metabolomics and proteomics in exploring the blood enriching mechanism of DBD in hemorrhagic anemia ratsSection 1.Study on the blood enriching mechanism of DBD in thymus and spleen of hemorrhagic anemia rats by an organ metabolomics approachThe previous studies had indicated that thymus and spleen were diseased in hemorrhagic anemia rat,and the lesions were significantly alleviated after administrated DBD.Thus,a non-target metabolomics approach was used to explore the treatment mechanism.Principal component analysis(PCA)was used to obtain the clustering trends of thymus and spleen metabolism profiles both in positive and negative modes.The results showed that the OPLS-DA score plots of thymus and spleen between normal and hemorrhagic anemia rats both in positive and negative modes were separated obviously.Furthermore,with the relative distances from blood deficiency group and DBD group to control group in the PLS-DA score plot,we suggested that after administrated with DBD,the situation of anemia was alleviated,and the biochemical perturbation was called back to normal.Combined with VIP scores in Loading plots,database and standards,a total of 10 endogenous biomarkers in thymus samples and 9 endogenous biomarkers in spleen samples were finally identified.The main endogenous biomarkers in thymus samples were lysophosphatidylcholines.Compared to normal group,the lysophosphatidylcholines(including LysoPC(16:0),LysoPC(18:2(9Z,12Z)),LysoPC(18:1(9Z)),LysoPC(18:0)and LysoPC(15:0)),indoxyl sulfate,D-Glucurono-6,3-lactone and cholic acid in model group were up-regulated(P<0.01),however,indoleacrylic acid and arachidonic acid were significantly down-regulated(P<0.01).In spleen samples,amino acids(including D-leucine,L-methionine and L-tyrosine),nucleosides(including thymine and xanthine)were the main biomarkers.Compared to normal group,amino acids,nucleosides and indoleacrylic acid in model group were down-regulated(P<0.05;P<0.01),retinyl ester,LysoPE(16:0/0:0)and prostaglandin El were up-regulated(P<0.01).The up-regulation of lysophosphatidylcholine and arachidonic acid in thymus suggested that thymus atrophy might be caused by thymocyte apoptosis due to excessive oxidative stress;the down regulation of nucleosides and amino acids in spleen would lead to the decrease of ATP by decreasing the mitochondrial quality,mitochondrial protein expression and mitochondrial respiration.After treatment with DBD,these differential biomarkers were significantly reversed,indicating that DBD could inhibit excessively oxidative stress and promote energy metabolismSection 2.Study on the blood enriching mechanism of DBD in thymus and spleen of hemorrhagic anemia rats by an organ proteomics approachIn this section,a Label-Free approach was used to explore the mechanism of protein level in thymus and spleen of hemorrhagic anemia rats,PEAKS 8.5 software and DAVID Webserver were employed to identify the differential proteins and GO function enrichment analysis respectively.Furthermore,the intervention effects of DBD was also studied.After the intervention of DBD,there were only 41 differential proteins in thymus,most of the proteins were down regulated in anemia rat,mainly including ribosome proteins,myeloperoxidase(Mpo),ceruloplasmin(Cp),hemopexin(Hpx),etc.Some other proteins,such as legumain(Lgmn),enoyl-CoA hydratase 1(Ech1),reactive intermediate imine deaminase A homolog(Rida),etc were up regulated.The total 41 differentially expressed proteins were up-loaded to DAVID to perform GO enrichment analysis.The results indicated that DBD could regulate oxygen transport,cellular iron ion homeostasis and response to oxidative stress via oxygen binding,peroxidase activity and heme binding in ribosome,extracellular space and so on.24 differential proteins were identified in spleen,including downed-regulated transferrin(Tf),NADH dehydrogenase(ubiquinone)Fe-S protein(Ndufs3),fatty acid binding protein 4(Fabp4),etc and up-regulated complement C3(C3),complement C3d receptor 2(Cr2),and mast cell protease 10(Mcpt10),etc in anemia rats.GO enrichment analysis results showed that DBD could promote the spleen mitochondrial complex biosynthesis,and ultimately accelerate oxidative phosphorylation to provide ATP.The results were consistent with those in metabolomicsSection 3.Integration analysis of phenotypes,metabolomics and proteomics and the effects of DBD on energy metabolismFunctional analysis results of differential biomarkers and proteins were all indicated that the thymus atrophy was related to the excessively oxidative stress of thymocytes,and the decline of spleen function was mainly related to the decline of mitochondrial function.For further explored the relationship among phenotypes,biomarkers and proteins,Pearson correlation matrix was applied,the results showed that phenotype key indexes were strong positively associated with most of the proteins and biomarkers,especially with nucleosides,amino acids,Fabp4,Decrl and Ndufs3.Indoleacrylic acid was increased and strong correlated with most of proteins both in thymus and spleen of anemia rats.Indoleacrylic acid was the metabolite of tryptophan,which had been reported had the effects of anti-inflammatory,it might be a new biomarker to hemorrhagic anemia.DBD was validated to up-regulate Mpo,Hbb and Cp levels and down-regulate Ca2+level in thymus,as well as up-regulate Fabp4,Ndufs3,Tf,Decrl and ATP levels in spleen.Through summarizing the literatures,the relationships between differential biomarkers and proteins were studied to simulate the biological process in thymus and spleen,respectively.In thymus,after modeling,the enhanced lipid metabolism decomposed lysophosphatidylcholines(LPC)and arachidonic acid,LPC could promote the formation of NOS,the up-regulation of Ca"+level and the occurrence of Fenton reaction in mitochondria,all of which would induce apoptosis by promoting oxidative stress.In addition,the low-expressed Mpo,Hgb,Hpx and Cp further promoted the development of oxidative stress.In spleen,after modeling,the down-regulated L-methionine,D-leucine,Fabp4 and Decrl inhibited the TCA cycle,and the down-regulated thymidine,xanthine and Ndufs3 inhibited the synthesis of mitochondrial complexes,the down-regulated Tf inhibited the transport of Fe3+,and mitochondrial complexes and Fe2+were the keyes of mitochondrial oxidative phosphorylation.The key protein levels were showed the same trends with proteomics results,and DBD could down-regulated the Ca2+level in thymus and up-regulated the ATP level in spleen.According to the results of this study,DBD could increase thymus function by anti-oxidative stress and reducing intracellular Ca2+level,and promote ATP production in spleen to provide energy to anemia rat.Chapter 4.Studies on blood enrichment and synergistic antitumor mechanismeffects of DBD combined with Fe and EPO in colon cancer-related anemia(CCRA)miceSection 1.Studies of blood enrichment effects of DBD combined with Fe and EPO in colon cancer-related anemia(CCRA)miceCancer-related anemia(CRA)was due to multiple etiologies,including chemotherapy-induced myelosuppression,blood loss,tumor as well as other factors.Oral or intravenous iron injection,subcutaneous injection of EPO and intravenous blood transfusion were commonly used in the treatment of CRA,attractively,in recent years,TCM had been widely used in CRA.Thus,in this section,CCRA model was induced by subcutaneous inoculation of CT-26 and i.p.with oxaliplatin.Fe,DBD and EPO were all applied to treat CCRA mice at the same dosage and different compatibility,and the evaluation indexes mainly included body weight,tumor volume,peripheral blood indexes,organ indexes,pathological analysis.Compared with C group,P and treatment groups showed lower body weight,after minus tumor weight,ED group showed the lowest body weight.Compared with A group,the tumor volumes in other groups were significant decreased,while the tumor volume of E,ED and DF groups was the largest,followed by P and EDF groups,and finally D-H and D-L groups.Only ED and EDF groups showed significant effects in every peripheral blood index.All of the treatment groups could decrease spleen index and had no effect on liver index.EDF and DF groups showed no effect on thymus index,and D-H and D-L groups showed no effect on kidney index.The above results suggested that only EDF group and ED group showed significantly blood enrichment effect,and ED group showed better,however,ED group showed larger tumor volume than P group and EDF group.Section 2.Studies on colonic contents microbiota modulation of DBD combined with Fe and EPO in CCRA miceThe bacteria that colonized the mammalian gut played a pivotal role in tumorigenesis and the response to therapy in classic mouse models of colon cancer.Iron was critical for the replication and survival of almost all bacteria,so in this section,changes of colonic contents microbiota in C,P,ED and EDF groups were detected with Illumina MiSeq sequencing.?-diversity indexes of Genus level showed that the richness,diversity and evenness of ED group were all lower than the other groups,however,the situation in EDF group was opposite.?-diversity showed that C group and P group was separated obviously,indicating that the gut microbial beta diversity was wrecked in CCRA mice.EDF group and ED group were closer to the C group in distance,and EDF group showed shorter distances,which indicated that EDF and ED could improve the flora imbalance and EDF showed better effect.The sequencing results of gut microbiota showed that compared to P group,the relative abundances of Lachnospiraceae and opportunistic pathogen(Odoribacter)in ED and EDF groups were decreased,which indicated that these two bacteria might be related to CCRA.Interestingly,EDF also decreased the relative abundances of cancer-related bacteria(Helicobacter,Lactococcus,Alloprevotella)and imbalance-inducing bacteria(Escherichia-Shigella and Parabacteroides)and increased the relative abundances of butyrate-producing bacteria(Ruminococcaceae UCG-014),however,ED showed the opposite effects to EDF,this might be the reason of the smaller tumor volume in EDF group.Section 3.Study on mechanism of DBD combined with Fe and EPO in intestinal contents of CCRA mice by a metabolomics approachThe non-target metabolomics was used to identify differential endogenous biomarkers in colonic contents in this section,which aimed to find out the blood enrichment and anti-tumor mechanism of EDF group.The OPLS-DA score plots between C and P groups both in positive and negative modes were separated obviously and clustered respectively.Furthermore,with the relative distances from P,ED and EDF groups to C group in the PLS-DA score plot,we suggested that endogenous disturbance in EDF group was the closest to that of C group.Finally,14 differential biomarkers were identified.After modeling,urobilinogen,aminoethoxyacetic acid,phytosphingosine,sphingosine,sulfolithocholylglycine,lysoPE(0:0/15:0),lithocholic acid,1-stearoylglycerophosphoglycerol,tauroursodeoxycholic acid,arachidonic acid and cholesterol sulfate were up-regulated,and L-tyrosine and nutriacholic acid were down-regulated.Urobilinogen,sphingosines,lithocholic acid,tauroursodeoxycholic acid,sulfolithocholylglycine and nutriacholic acid in ED group were up-regulated more obviously.The results indicated that they might be the key biomarkers which related to tumor growth.The relative intensities of tyrosine and arachidonic acid in ED and EDF were both decreased with same trends,which indicated that they might be related to CCRA.Correlation analysis showed that sphingosine-1-phosphate was positively correlated with Helicobacter.Pathway analysis showed that the main pathways included phenylalanine,tyrosine and tryptophan biosynthesis,arachidonic acid metabolism,tyrosine metabolism and sphingolipid metabolism,and the increased tumor volume in ED group was mainly due to the increase of anti oxidative stress ability of tumor cells after sphingolipid metabolism dysregulation.