Preliminary Study On Potential Pharmacodynamic Components And Pathways Of Danggui Buxue Decoction In Promoting Erythropoiesis

Objective: To study the potential pharmacodynamic components of Danggui Buxue Decoction in promoting erythropoiesis,and to explore the pathway of Danggui Buxue Decoction in promoting erythropoiesis.Method:First.The Pharmacodynamics of DBT in promoting erythropoiesis was studied.1)Two factors and three levels were designed,different concentration(0.2 ug/mL,0.1 ug/mL,0.067 ug/mL)and different incubation time(15 min,30 min,60 min).Flow cytometry was used to measure the ratio of reticulocytes to optimize the method of reticulocyte test.2)The mice were divided into control group(saline),high dose group(10 g/kg),medium dose group(5 g/kg)and low dose group(0.1 g/kg).Five mice in each group were given intragastric and intraperitoneal injection respectively.The ratio of reticulocytes was by intragastric and intraperitoneal injection,and the administration mode,dosage and time point were determined by analyzing the change of the ratio of reticulocytes.3)CD71-FITC and Ter119-PE labeled bone marrow single cell suspension were used to verify the effect of DBT on early,middle and late erythropoietic related cells in bone marrow.4)CFU-E medium was used to culture BMSCs.After 48 hours,the number of CFU-E clones was counted.5)BFU-E medium was used to culture BMSCs,After 7 days,the number of CFU-E clones was counted to verify the ability of DBT to differentiate red blood cells.Second.The Potential Pharmacodynamic Components of DBT for Erythropoiesis was studied.1)The preparation method of autolo gous serum of mice and the maximum amount of serum added was optimized by MTT test.2)After the last administration,drug-containing serum was prepared at different time points according to the preparation method of autologous serum.3)The serum containing drugs were divided into small molecular group of Chinese medicine metabolism and large molecular group of protein by 3K ultrafiltration tube,and the effect of these on BMSCs culture was observed were by cell proliferation experiment.4)Blood Input Components and Endogenous Components in Serum Containing Drugs at Different Time Points was analysized by Metabolites.5)The potential pharmacodynamic components of DBT for erythropoiesis was searched by spectral-activity relationship and correlation analysis.Thrid.The target protein of DBT for Erythropoiesis was studied.1)The target proteins and ac tion pathways of DBT promoting Erythropoiesis was studied by Proteomic from the protein level.2)The expression of related proteins was verify by Elisa.Fouth.The extramedullary hematopoietic pathway of spleen was studied.1)The effect of DBT on spleen of mice was ananlyzed by HE staining.2)The effect of DBT on differentiation of erythroid progenitor cells into mature erythrocytes in spleen of mice was verified by CFU-E and BFU-E experiments.3)The effects of DBT on erythroid-related cells in spleen of mice was detected by Flow cytomery.4)The effects of DBT on gene expression in spleen of mice was analyzed by transcriptome assay,and the pathway of extramedullary hematopoiesis in DBT spleen was predicted.5)The mechanism of DBT promoting extramedullary hematopoiesis in spleen of mice at gene level was verified by Q-PCR.Result:The second chapter: The Pharmacodynamics of DBT in promoting erythropoiesis was studied.1)The proportion of reticulocytes in thiazole orange with final concentration of 0.1 ug/mL was relatively stable when incubated in dark for 30 minutes at 37?.2)Compared with intraperitoneal administration,the proportion of reticulocytes in control group of intraperitoneal injection group fluctuated less and was more stable.3)After intraperitoneal injection of DBT,the percentage of reticulocytes in 10 g/kg and 5 g/kg groups increased with time,but there was lethality in 10 g/kg mice.4)The proportion of reticulocytes increased significantly after 14 days of continuous administration.5)After the last administration,the spleen index(5.22±0.4)in DBT group was significantly(p < 0.05)higher than that in control group(4.12 ±0.17).6)The proportion of erythrocyte-related cells increased significantly by flow cytometry,and the proportion of early erythrocyte-related cells increased significantly.The proportion of erythroid-related cells increased significantly in the middle stage,indicating that the early and intermediate erythroid-related cells may be potential targets for DBT early and intermediate erythroid-related cells may be potential targets for DBT.7)The number of CFU-E and BFU-E cells increased significantly in BMSCs culture,indicating that DBT may promote erythroid progenitor cell differentiation into mature erythrocyte.The third chapter: The Potential Pharmacodynamic Components of DBT for Erythropoiesis was studied.1)No anticoagulant and inactivation of serum are more suitable for BMSCs culture in vitro.2)5% autologous serum is the best growth state for BMSCs culture in vitro.3)After freeze-dried serum,20% serum freeze-dried powder is the best growth state for BMSCs culture in vitro.4)When BMSCs are cultured with medicated serum at different time points,the pharmacodynamic activity of small metabolic molecules in medicated serum follows 30,60 and 90 minutes.The pharmacodynamic activity of serum small molecule obtained in 60 minutes was significantly increased compared with the control group,while the pharmacodynamic activity of serum small molecule obtained in 120 minutes showed a downward trend,followed by an upward trend.The cell activity of serum containing drugs in 180 minutes was significantly different from that in 120 minutes.5)Metabonomics based on UPLC-MS was used to study all metabolites in drug-containing serum.The correlation between metabolites and in vitro cell pharmacodynamics was analyzed.Seven DBT blood-entry components and 15 endogenous components with high correlation with pharmacodynamics were obtained.6)The correlation between DBT blood-entry components and endogenous metabolites and the profiles of metabolic pathways of endogenous components showed that DBT might pass through glutamic acid,TCA cycle,glutamine,arginine,tryptophan and threonine promotes the formation of erythroid-related cells,and glutamic acid is the node molecule of metabolic pathway.The fourth chapter,The target protein of DBT for Erythropoiesis was studied.1)Metabolic macromolecules of traditional Chinese medicine promote the proliferation of BMSCs.2)Proteome analysis showed that 698 proteins were identified in the serum of the drug-treated group and the control group,including 243 differential proteins,190 up-regulated differential proteins and 53 down-regulated differential proteins.3)There are 20 potential differential proteins to promote erythrocyte production by participating in the homeostasis of bone marrow hematopoietic microenvironment by filtering different differential proteins,mainly for Regulation of actin cytoskeleton,focal adhesion,Adherens junction,Cell adhesion molecules(CAM),Hematopoietic related proteins.4)The expression of Ltf,Vinculin and CD14 increased by Elisa.The fifth chapter,The extramedullary hematopoietic pathway of spleen was studied.1)HE staining of spleen slices showed an increase in the number of splenic granulocytes in the administration group.2)The number of CFU-E and BFU-E clones in the spleen of mice in the administration group increased significantly.3)The number and proportion of erythrocyte-related cells in the spleen of mice in the administration group increased,suggesting that DBT may promote extramedullary hematopoiesis in the spleen.4)Splenic extramedullary hematopoiesis is mainly related to chemotaxis,regulation,production of cytokines and Jak-STAT pathway.which is predicted by the resulte of spleen transcriptome.5)The CXCL2,CCR1,CXCL1,IL-1a,PTGS2,Pim1,IL-10,CXCR2,SOCS3,IL-1b gene expression increased significantly by Q-PCR test.Conclusion:DBT has the pharmacological effect of promoting erythropoiesis.Firstly,The potential pharmacodynamic components of DBT promoting erythropoiesis are RHA,TPA,TO,MEOA,PA,CDVD3 and DDG.The seven potential DBT blood-feeding components promote the formation of erythroid-related cells by promoting glutamate metabolism and further affecting metabolites in other pathways;Secondly,DBT indirectly promotes erythrocyte production by promoting the expression of target proteins LTF,Vinculin and CD14 and triggering the expression of regulatory of actin cytoskeleton,focal adhesion,Adherens junction,Cell adhesion molecules(CAM)and Hematopoietic related pathways.Thirdly,DBT can promote erythrocyte production by activating chemotaxis,production of cytokines and gene expression related to Jak-STAT pathway to trigger bypass pathway-extramedullary hematopoiesis of spleen.