Based On Data Mining To Explore The Mechanism Of Coordinating Hematopoiesis By Multiple Organs Of Caulis Spatholobi

Objective:Objective to explore the targets and pathways of the water extract of Caulis Spatholobi cooperating with multiple organs to regulate hematopoiesis.Methods:First.The data mining of the regulation of hematopoiesis by Caulis spatholobi and its efficacy verification.(1)Quality control of the water extract of Caulis spatholobi.Catechin was used as the Chinese medicine marker of Spatholobus spatholobi,and the quality standard for the water extract of Spatholobus spatholobi was constructed using the HPLC spectrum of Chinese medicine;(2)Integrating multiple databases and literature search to find chemical components of Caulis Spatholobi,predicting drug targets based on pharmacophore model of chemical components,and establishing target database.The drug targets and anemia disease targets were intersected to obtain the targets related to anemia disease.The target organs were obtained by enrichment analysis of targets,and then the pathways and targets related to hematopoiesis were obtained by enrichment analysis based on KEGG database.The above targets were analyzed by PPI to find the key targets.Autodock Vina was used to analyze the binding ability of key targets and related pharmacodynamic components.(3)The cell proliferation experiment optimized the cell culture density and the water extract concentration of the chicken blood vine when cultured in vitro;the bone marrow cells were labeled with CD71 and TER119,and the effects of the aqueous extract of the chicken stem stem on the red blood line related cells were detected by flow cytometry;BFU-E and CFU-E were used to detect the proliferation and differentiation ability of the erythrocytes.Second.Objective to study the effect of the water extract of Caulis spatholobi on hematopoietic regulation by targeting bone marrow cells.(1)Cell proliferation assay was used to detect the effect of the water extract of Caulis spatholobi on the proliferation of bone marrow stromal cells,flow cytometry was used to detect the cell cycle of bone marrow stromal cells cultured with the water extract of Caulis spatholobi,and Q-PCR was used to detect the changes of cell cycle related genes.(2)Use the migration experiment to explore the migration effect of the water extract of Caulis spatholobi on bone marrow stromal cells;(3)Q-PCR and Elisa detect the related genes that promote cell migration from the gene and protein levels,respectively.Third.The water extract of Caulis Spatholobi affects erythroid-related cells by regulating cell apoptosis.(1)Use Cluster Profiler package and Clue GO package to further GO function annotation and KEGG enrichment analysis;(2)Use flow cytometry to detect the influence of the water extract of Caulis Spatholobi on erythroid cell apoptosis;3)Use Q-PCR,Elisa And Western Blot,to verify apoptosis-related genes and proteins from the gene and protein levels.Fourth.To study the role of extramedullary hematopoiesis pathway.(1)Further functional annotation and KEGG enrichment analysis of go were carried out by using clusterprofiler package and clustergo package;(2)Flow cytometry was used to detect the proportion of reticulocytes in the peripheral blood of mice,so as to determine the efficacy of the water extract of Caulis Spatholobi and determine the sampling time;(3)The body weight and organ index of mice were analyzed;(4)Flow cytometry was used to detect the effect of the water extract of Caulis Spatholobi on erythroid related cells in mice kidney;(5)Q-PCR was used to verify the mechanism of promoting extramedullary hematopoiesis of mouse kidney by aqueous extract of Caulis Spatholobi.Result:First.Data mining analysis and pharmacodynamic verification of Caulis Spatholobi.(1)Objective to study the quality control of the water extract of Caulis Spatholobi.Using Catechin as the traditional Chinese medicine marker of the water extract of Caulis Spatholobi,the chromatogram of the water extract of Caulis Spatholobi was analyzed by high performance liquid chromatography(HPLC).The retention time of catechin was 23.38 min,and the Catechin had the highest absorption value at 279.5 nm.Through comparative analysis,this substance also existed in spatholobus suberectus water extract.Each 250 mg the water extract of Caulis Spatholobi contains 0.232μg of Catechin;(2)There were 1614related targets of sinomenis,among which 805 targets were related to anemia.Through the enrichment of target organs and target cells,the main target organs of Caulis Spatholobi are bone marrow,kidney,hematopoietic stem cells,centrioles,platelets and cd4-cd28+/-T cells in blood;the KEGG enrichment analysis of these genes was carried out by Clusterprofiler package.It was found that there were 9 hematopoietic related pathways in bone marrow,10 blood related pathways and 4 renal related pathways;Molecular docking showed that genistin and PTGS2,ononin and PTGS2,(+)-catechin and PTGS2,genistin and MMP9,olmelin and MMP9 had the highest binding power;(3)The proliferation experiment optimized the culture density of bone marrow stromal cells to be 4.0*10~6～2.0*10~6 cells/m L and the best concentration of 0.25 mg/m L～0.5 mg/m L in vitro;Flow cytometry was used to detect the erythroid related cells of bone marrow stromal cells.Compared with the control group,the percentage of erythroblasts in the extract group was significantly increased(21.57±1.38)%compared with the control group(14.17±0.91)%(P<0.05);The percentage of erythroblasts(74.77±1.77)%was significantly higher than that of the control group(67.07±1.13)%(P<0.05),the results showed that the water extract of Caulis Spatholobi could promote the development and maturation of erythroid related cells to early and middle erythroblasts;(3)In BFU-E experiment,the number of cell clones in the water extract of Caulis Spatholobi group(0.25 mg/m L,0.5 mg/m L)(121.00±5.60,109.70±3.00)was significantly higher than that in the control group(68.67±1.80)(P<0.001);In CFU-E experiment,the number of cell clones in the water extract of Caulis Spatholobi(0.25 mg/m L,0.5 mg/m L)was 189.70±8.70,295.70±13.12,which was significantly higher than that in the control group(133.70±5.40)(P<0.01),these results indicate that the water extract of Caulis Spatholobi can promote the proliferation and differentiation of hematopoietic progenitor cells to downstream erythroid cells.Second.Objective to study the effect of the water extract of Caulis Spatholobi on hematopoietic regulation by targeting bone marrow cells.(1)The water extract of Caulis Spatholobi could increase the number of bone marrow stromal cells and prolong the culture time in vitro;Flow cytometry showed that the water extract of Caulis Spatholobi could promote the cells to enter the proliferation cycle;Q-PCR confirmed that MYC,MCL1,TLR4,TLR2 and CASP3 were up-regulated(P<0.05);(2)Compared with the control group,the cell migration rate in the water extract group increased significantly(P<0.01),and with the increase of time,the cell migration rate in the water extract group increased gradually(P<0.0001),the water extract of Caulis Spatholobi promoted the migration of bone marrow stromal cells;The expression of IL10,VEGFA,VCAM1 and CCL2 was significantly increased by Q-PCR(P<0.05);The expressions of IL10 protein(P<0.005)and VEGF protein(P<0.05)were detected by ELISA.The expressions of IL10 and VEGF protein were significantly increased,these results indicate that IL10 and VEGF are potential targets of the water extract of Caulis Spatholobi to promote the migration of bone marrow stromal cells.Third.The water extract of Caulis Spatholobi influences erythropoiesis by regulating apoptosis.(1)Target cells enriched in the blood include hematopoietic stem cells,centrioles,platelets and CD4-CD28+/-T cells,and visually display target cells and their relative targets;(2)The Clusterprofiler package was used to annotate the functions of these genes,and the Clue GO package was used to analyze KEGG.It was found that these genes were related to cell proliferation,apoptosis and other functions.(3)Flow cytometry showed that the early apoptosis rate of erythroid related cells decreased significantly in the water extract group(P<0.05);(4)Q-PCR experiments verified apoptosis-related genes,such as BCL-2.The expression of BAX,BCL-XL,CXCR2,CXCR1 and other genes increased significantly;(5)Elisa experiment detected TNF-αprotein(P<0.005),and its protein expression increased significantly;WB experiment verified BCL-2 and BAX,after administration,the ratio of BCL-2/BAX was significantly increased(P<0.005).Fourth.The study of the pathway of extramedullary hematopoiesis in kidney.(1)The gene enriched in kidney was annotated by Clusterprofiler package and KEGG analysis of Clue GO package.It was found that these genes were related to cell proliferation,adhesion,apoptosis and other functions;(2)After 15 days of continuous administration,reticulocytes in peripheral blood of mice were significantly increased by flow cytometry(P<0.05);(3)The body weight of mice in the water extract group(40 g/kg,10.8 g/kg,1 g/kg)tended to increase significantly,and the kidney index was significantly higher than that in the normal saline group(P<0.05),but the spleen index was not significant;(4)The ratio of early and middle erythroblasts in the kidney of mice in the water extract of Caulis Spatholobi group(40 g/kg,10.8 g/kg,1 g/kg)was increased(P<0.05),indicating that the water extract of Caulis spatholobi can promote renal extramedullary hematopoiesis;(5)Q-PCR verified that the genes related to extramedullary hematopoiesis in the mouse kidney,MMP9,FOS,PTGS2,CXCL8(P<0.05),were all significantly expressed at the genetic level.Conclusion:Data mining and analysis of Caulis Spatholobi,predicting that Caulis spatholobi can coordinately regulate related target genes and pathways through bone marrow,target cells in blood,and kidney multiple organs to promote hematopoiesis.(1).The water extract of Caulis Spatholobi regulates hematopoiesis by regulating the expression of VCAM-1,IL10 and VEGFA in the bone marrow;and regulates the cell cycle through CCL2,TLR4 and CXCR4 and other genes to regulate hematopoiesis;(2).The water extract of Caulis Spatholobi passes through the blood the target cells indirectly affect the hematopoietic microenvironment and inhibit apoptosis-related cytokines,such as BCL-2,BAX,BCL-XL,CXCR2,etc.(3).The water extract of Caulis spatholobi can trigger TNF,IL-17 pathway and CXCR as the axis through genes such as MMP9,FOS,PTGS2,CXCL8 and other related chemokines to activate the pathway-renal extramedullary hematopoiesis to promote the production of erythroid cells.