Preliminary Study On Erythropoiesis Promoting Pathway Of Danggui Buxue Tang Based On Network Pharmacology

Objective: To study the effect of Danggui Buxue Tang(DBT)on erythropoiesis.Methods:First.Study on the network pharmacology of DBT.1)Based on literature research and TCM System Pharmacology database and analysis platform(tcmsp,http://lsp.nwu.edu.cn/,version 2.3),obtain the chemical components of Angelica and Astragalus;2)According to the two indexes of bioavailability(OB)≥ 30% and drug like(DL)≥ 0.18,the related effective components were screened.At the same time,it added the ingredients which did not meet the screening criteria but had high content or significant activity in DBT;3)Using stitch(http://stick.embl.de/),switchtargetprediction(switch,http://swisstarge tprediction.ch/),similarity ensemble approach(sea,http://sea.bkslab.org/)to obtain the target genes related to the related pharmacodynamic components;4)Using cluster profiler package to annotate the diseases related to the target gene and find the genes related to the diseases of the blood system.Based on the KEGG、GO database,using cluster profiler R package to annotate the functions.Second.The role of bone marrow stromal cells in the regulation of erythropoiesis by DBT.1)The concentration of DBT was optimized by MTT;2)CD71 and Ter119 were used to label bone marrow cells,and flow cytometry was used to detect the effect of DBT on erythroid related cells;3)BFU-E and CFU-E were used to detect the proliferation and differentiation of hematopoietic progenitor cells;4)The role of bone marrow stromal cells in cobblestone formation was detected;5)The effect of DBT on gene expression of mouse bone marrow stromal cells was analyzed from gene level by transcriptome,and the way of bone marrow stromal cells to play a role was predicted.Meanwhile,Q-PCR was used to verify.Third.DBT promotes the secretion of cytokines by bone marrow stromal cells.1)To optimize the preparation method and the optimal concentration of DBT conditioned medium;2)Using 3K ultrafiltration tube,the conditioned medium was divided into macromolecular group and micromolecule group.SDS-PAGE was used for quality control.At the same time,cell proliferation experiment was used to detect the effect of macromolecular and micromolecule on the culture of Bone marrow stromal cells in vitro;3)Q-PCR was used to detect the gene expression changes of related cytokines,and the qualified cytokines were screened out.4)Flow cytometry was used to detect the effect of conditioned medium macromolecule on the apoptosis of erythroid related cells;5)Q-PCR and Western blot were used to detect the effect of conditioned medium macromolecule on apoptosis related genes and proteins.Fourth.Study on the effect of DBT on the formation of erythroblastic island.1)The proportion of reticulocytes in the peripheral blood of mice was detected by flow cytometry,so as to judge the efficacy of DBT and determine the time of sampling;2)Flow cytometry was used to detect the effect of DBT on the proportion of basophilic erythroblasts,Polychromatic erythroblasts and orthochromatic erythroblasts in erythroid related cells of mice;3)BFU-E and CFU-E were used to detect the proliferation and differentiation of hematopoietic progenitor cells;4)HE staining and immunofluorescence were used to detect the effect of DBT on erythroblast island of mice;5)Q-PCR was used to detect the changes of the genes related to the erythroblastic island from the gene level.Result:First.Network pharmacology research of DBT.1)A total of 212 chemical compositions were obtained,and 17 potential medicinal ingredients of DBT were screened based on the two indicators of OB≥30% and DL≥0.18.Then 15 kinds of medicinal ingredients that do not meet the screening criteria but have high content or significant activity Ingredients,a total of 32 medicinal ingredients;2)TCMSP,STITCH,SEA,and SWISS were used to predict the targets of the 32 active ingredients,and 1217 related genes were found;3)Using the Cluster Profiler package to annotate the functions of these genes,it was found that there are 81 genes related to hematopoietic diseases;4)Through GO and KEGG annotation of hematopoietic related genes by cluster profiler package,we found that these genes are related to monocyte proliferation,adhesion,apoptosis,Cytokine activity,PI3K-AKT pathway and other functions.Second.Study on the effect of bone marrow stromal cells in the regulation of erythrocytes production by DBT.1)In vitro experiments showed that there was a dose-response relationship between the number of bone marrow stromal cells and the dosage of DBT.Compared with the control group,the number of bone marrow stromal cells increased significantly at 0.5,1,2 and 4 mg/m L,the number of bone marrow stromal cells was the most at 4 mg/m L,and decreased at 8 mg/m L showing certain side effects.In order to avoid side effects,the subsequent concentration of 2 mg/m L;2)Flow cytometry showed that compared with the control group,the proportion of DBT in basophilic erythroblasts and polychromatic erythroblasts increased significantly(P=0.0016,P=0.0319),indicating that DBT may promote the development and maturation of erythrocytes in basophilic erythroblasts and Polychromatic erythroblasts;3)In BFU-E and CFU-E experiments,the number of cell clones in the group of DBT increased significantly(P=0.0067,P=0.0041),which indicated that DBT could promote the proliferation and differentiation of hematopoietic progenitor cells to the downstream erythrocytes;4)By clone formation experiment,bone marrow cells were co cultured with DBT in the presence or absence of bone marrow stromal cells.It was found that when co cultured with bone marrow stromal cells,the number of cell clones increased significantly,indicating that bone marrow stromal cells play an important role;5)The results of transcriptome analysis showed that ECM receiver interaction,PI3K-AKT,focal adhesion and other pathways changed in DBT group compared with the control group.These pathways are related to adhesion,including COL6A3,TNN,THBS1,ITGA2 B,PI3K,AKT,CCND2 and so on.ECM-PI3K-AKT-CCND2 is common to network pharmacology analysis;6)Q-PCR experiments verified that the expression of COL6A3,TNN,THBS1,ITGA2 b,PI3K,AKT,CCND2 and other genes was significantly increased.Third.Study on the promotion of cytokines secreted by bone marrow stromal cells by DBT.1)Compared with the control group,the number of bone marrow stromal cells in the conditioned medium group was significantly increased(P=0.0387);2)SDS-PAGE experiments ensure that large and small molecules can be separated after centrifugation in 3K ultrafiltration tube.Compared with the control group,the number of bone marrow stromal cells in the conditioned medium macromolecule group increased significantly(P=0.0085);3)The proportion of early apoptosis of erythroid related cells in the conditioned medium macromolecule group decreased significantly(P=0.047);4)The genes of FGF,MYDGF,FLT3 and SCF were detected by Q-PCR.It was found that the expression of FGF,MYDGF and FLT3 was significantly up-regulated;5)The apoptosis related genes were confirmed by Q-PCR.In conditioned medium macromolecule group,BCL-2 and BCL-x L were significantly upregulated,BAX and BAD were significantly down regulated.WB experiment confirmed that BCL-2 and BAX,after administration,the proportion of BCL-2/BAX was significantly up-regulated(P=0.0148).Fourth.Study on the effect of DBT on the formation of erythroblastic island.1)After continuous administration of DBT for 15 days,reticulocytes were significantly increased in peripheral blood of mice(P = 0.039);2)The proportion of erythroblasts in the early and middle stage increased significantly(P = 0.017,P = 0.01);3)In BFU-E and CFU-E experiments,the number of cell clone increased significantly,which indicated that hematopoietic progenitor cells might be the potential target of DBT;4)HE staining of femur and immunofluorescence detection showed that the number of erythroblastic islands in DBT group increased significantly(P<0.0001);5)Q-PCR was used to detect VCAM-1,ITGA5,ICAM-4,ITGA4,ITGB1,EMP and other genes,among which the expressions of VCAM-1 and ITGB1 were significantly up-regulated,and they were closely related to the adhesion of macrophages and erythrocytes in the erythroblast island.Conclusion:After the network pharmacology analysis of DBT,it is predicted that the hematopoietic related genes are related to monocyte proliferation,adhesion,apoptosis,PI3K-AKT pathway and other functions.DBT indirectly promotes erythropoiesis through bone marrow stromal cells,and it works through COL6A3,TNN,THBS1,ITGA2 B,PI3K,AKT,CCND2 and other genes in ECM-PI3K-AKT pathway.DBT Tang can inhibit apoptosis by promoting marrow stromal cells to secrete myelogenous growth factor.DBT Tang can promote the formation of erythropoietic islands in the bone marrow of mice,which provides a good environment for bone marrow hematopoiesis.