Based On Hematopoietic Regulatory Network And Data Mining To Explore The Synergistic Mechanism Of Carica Papaya Leaf In The Treatment Of Anemia

Objective: From a holistic point of view,using data mining to explore the mechanism of Carica papaya leaf(CPL)multi-component synergy,through multi-target and multi-organ treatment of different types of anemia.Methods:First.Data mining of chemical compounds and targets of CPL and establishment of anemia classification.1)Pub Med database and CNKI database were used to search CPL chemical compounds and construct chemical compounds database;2)The SMILES structure and "sdf" format of the compounds were obtained from Pub Chem database,and the compounds were clustered according to their structural similarity using Rcpi R package;3)The target of each chemical compound was obtained from TCMSP,Pharmmapper,Swiss Target Prediction,SEA Search Server,STITCH and Pub Chem database;4)The targets of the six types of databases were imported into Metascape website for functional annotation and classification;5)Dose R package was used to perform Disease enrichment analysis on the compound targets based on the Disease Ontology and Dis Ge NET database.The screening standard was p<0.05,and the diseases were classified according to the anemia pathology,and the related targets of CPL in the treatment of anemia were obtained.Second.Analysis of key targets and pathways of CPL in the treatment of hemorrhagic anemia/aplastic anemia/sickle cell anemia.1)Through the cluster Profiler R package and based on the Cell Marker database,target organs were enriched by targets associated with anemia of CPL.The screening standard was p<0.05,and the targets enriched in bone marrow,blood,liver and kidney were obtained;2)The cluster Profiler R package was used to conduct pathway enrichment analysis of targets in bone marrow,blood,liver and kidney based on KEGG database.The screening standard was p<0.05.The pathways of various anemia in each organ were obtained,and the pathways related to hematopoiesis were retained,and the relationships among the pathways were established;3)The key targets and corresponding active compounds of CPL were obtained by PPI analysis and molecular docking of the related targets in the action pathways of each organ.Third.Effect of CPL on hematopoiesis based on the common pathways and key targets of three kinds of anemia in bone marrow.1)The target points of hemorrhagic anemia,aplastic anemia and sickle cell anemia in bone marrow were analyzed based on KEGG database respectively.The common pathways that can regulate three kinds of anemia at the same time were obtained.PPI analysis and molecule docking were performed to obtain the key targets and corresponding active compounds;2)The effects of different concentrations of CPL on the viability of bone marrow cells were detected in vitro,and then the optimal concentration of CPL was confirmed;3)Flow cytometry was used to detect the effect of CPL on the number and proportion of erythroid related cells in mouse bone marrow;Cell cycle assay was used to detect the effect of CPL on bone marrow cell cycle;4)Cell migration assay was used to detect the effect of CPL on the migration of bone marrow stromal cells;5)Real time fluorescent quantitative PCR was used to detect the expression of the selected key targets in bone marrow cells after CPL treatment.Result:First.Data mining of chemical compounds and targets of CPL and establishment of anemia classification.1)A total of 139 known chemical constituents of CPL were collected.Clustering is carried out according to structural similarity,which can be divided into six categories: fatty acids,small molecules,N compounds,esters,flavonoids and others;2)A total of 1868 CPL related targets were obtained based on six databases.The functional annotation classification of the targets was carried out,which were divided into14 categories,including transferases,hydrolases,oxidoreductases,transporters,G protein coupled receptors,transcription factors,transporter channels,CD markers,lyases,RAS pathway related proteins,ligases,isomerases,cytokines and others.3)The targets of CPL were enriched and classified into seven different types of anemia,including hemorrhagic anemia,aplastic anemia,sickle cell anemia,hemolytic anemia,thalassemia,iron deficiency anemia and macrocytic anemia.Second.Analysis of key targets and pathways of CPL in the treatment of hemorrhagic anemia.1)After enrichment in target organs to related targets for hemorrhagic anemia,27 bone marrow targets,82 blood targets,56 liver targets and 15 kidney targets were obtained;2)Through the enrichment of KEGG pathway,21,23,15 and 6hematopoietic related pathways were obtained;3)After PPI analysis and molecular docking,the key targets and active compounds of various organs for the treatment of hemorrhagic anemia were obtained,including CXCR4-Papain(Affinity=-6.9kcal/mol)and PECAM1-Ursolic acid(Affinity=-7.4kcal/mol)in bone marrow,VEGFA-Ursolic acid(Affinity=-7.3kcal/mol)and ICAM1-Hesperidin(Affinity=-11.3kcal/mol)in blood cell,STAT3-Pheophorbide A(Affinity=-8.7kcal/mol)and CASP3-Oleanolic acid(Affinity=-8.3kcal/mol)in liver,MMP9-Nicotiflorin(Affinity=-9.3kcal/mol),MMP9-Rutin(Affinity=-9.4kcal/mol)and CXCL8-Fisetin(Affinity=-7.3kcal/mol)in kidney.Third.Analysis of key targets and pathways of CPL in the treatment of aplastic anemia.1)After enrichment in target organs to related targets for aplastic anemia,29 bone marrow targets,40 blood targets,18 liver targets and 2 kidney targets were obtained;2)Through the enrichment of KEGG pathway,20,27,9 and 2 hematopoietic related pathways were obtained;3)After PPI analysis and molecular docking,the key targets and active compounds of various organs for the treatment of aplastic anemia were obtained,including IL10-Quercetin(Affinity=-7.0kcal/mol)and CXCR4-Papain(Affinity=-6.9kcal/mol)in bone marrow,VEGFA-Ursolic acid(Affinity=-7.3kcal/mol)and ICAM1-Hesperidin(Affinity=-11.3kcal/mol)in blood cell,CASP3-Oleanolic acid(Affinity=-8.3kcal/mol)and IFNGQuercetin(Affinity=-7.8kcal/mol)in liver,CA1-Nicotiflorin(Affinity=-8.7kcal/mol)in kidney.Fourth.Analysis of key targets and pathways of CPL in the treatment of sickle cell anemia.1)After enrichment in target organs to related targets for sickle cell anemia,20 bone marrow targets,32 blood targets,11 liver targets and 9 kidney targets were obtained;2)Through the enrichment of KEGG pathway,14,20,4 and 3hematopoietic related pathways were obtained;3)After PPI analysis and molecular docking,the key targets and active compounds of various organs for the treatment of sickle cell anemia were obtained,including IL10-Quercetin(Affinity=-7.0kcal/mol)and VEGFAUrsolic acid(Affinity=-7.3kcal/mol)in bone marrow,ICAM1-Hesperidin(Affinity=-11.3kcal/mol)and VCAM1-Hesperidin(Affinity=-7.7kcal/mol)in blood cell,IFNG-Quercetin(Affinity=-7.8kcal/mol),HMOX1-Oleanolic acid(Affinity=-9.0kcal/mol)and HMOX1-Ursolic acid(Affinity=-9.0kcal/mol)in liver,CXCL8-Fisetin(Affinity=-7.3kcal/mol)and PTGS2-Myricetin(Affinity=-10.3kcal/mol)in kidney.Fifth.Effect of CPL on hematopoiesis based on the common pathways and key targets of three kinds of anemia in bone marrow.1)Through the pathway enrichment of three kinds of anemia related target,47 common pathways located in bone marrow that can regulate three types of anemia at the same time were obtained,including PI3 K Akt,MAPK and other signaling pathways.Key targets and active compounds were obtained by PPI analysis and molecular docking,including VEGFA-Ursolic acid(Affinity=-7.3kcal/mol),IL10-Quercetin(Affinity=-7.0kcal/mol),PECAM1-Ursolic acid(Affinity=-7.4kcal/mol),CCL2-Quercetin(Affinity=-7.0kcal/mol),VCAM1-Hesperidin(Affinity=-7.7kcal/mol).2)The results of HPLC showed that catechin was used as the control substance for quality control,and CPL extract was used as the sample.The retention time and full wavelength scanning results of the two were consistent,indicating that there were catechins in CPL;3)The results of MTT assay showed that compared with the control group,CPL had a significant promoting effect on the proliferation of bone marrow cells when the treatment concentration was 1 mg/m L and the treatment time was 6 days(p=0.029);4)Flow cytometry results showed that CPL extract could increase the number and proportion of early erythroblasts in erythroid related cells in mice bone marrow(p=0.030),suggesting that CPL could promote hematopoiesis.However,the effect of CPL on bone marrow cell cycle was less(p=0.140,p=0.995);5)Cell migration assay showed that cell migration rate increased significantly when cultured with CPL at 1 mg/m L for 72h(p=0.010),indicating that CPL could promote the migration of bone marrow stromal cells;6)The expression changes of key targets were detected by q PCR.Compared with the control group,the expression of VEGFA increased significantly(p=0.030),indicating that CPL can affect the expression of VEGFA.Conclusion:1)The active substances of CPL mainly include Quercetin,Ursolic acid,Oleanolic acid,Hesperidin,Papain,Pheophorbide A,Nicotiflorin,Rutin,Fisetin and Myricetin.2)The key targets of CPL include VEGFA,VCAM1,ICAM1,CXCR4,CASP3,STAT3,CXCL8,IL10,PECAM1,IFNG,MMP9,HMOX1,PTGS2,CCL2 and CA1.3)The action pathways of CPL mainly include PI3K-Akt signaling pathway,MAPK signaling pathway,Focal adhesion,Cell adhesion molecules,HIF-1 signaling pathway,JAK-STAT signaling pathway,Cytokine-Cytokine receptor interaction,IL-17 signaling pathway and Nitrogen metabolism.4)CPL could promote the proliferation of bone marrow cells,increase the number and proportion of early erythroblasts,promote the migration of bone marrow stromal cells,and enhance the expression of VEGFA in bone marrow cells.