Adhesion Mechanism Of Danggui Buxue Tang On Bone Marrow Stromal Cells And Its Effect On Hematopoietic Function

Objective:To study the adhesion and mechanism of Danggui BuxueTang(DBT)on bone marrow stromal cells(BMSCs).To explore the ability of Danggui Buxue Tang to regulate hematopoiesis in vitro.Methods:First,Ferulic acid and fermononetin were used as Chinese traditional medicine markers of Angelica sinensis and Astragalus membranaceus,respectively,and the quality control standard of Danggui Buxue Tang was constructed by the fingerprint of Chinese medicine.Second.The optimum cell culture density and drug use concentration of bone marrow stromal cells(BMSCs)in vitro were optimized by MTT test.The effect of DBT on the culture of BMSCs in vitro was observed according to cell proliferation experiment,cell cycle and long-term culture in vitro.At the same time,the adhesion and migration of DBT to BMSCs cells was investigated by adhesion and migration experiments.The expression of adhesion protein and the change of cytoskeleton can be observed by immunofluorescence staining.Third.The transcriptional test reflects the gene expression of DBT in BMSCs cells from the gene level,to predict the mechanism of BMSCs’adhesion.The effect of the Focal Adhesion signaling pathway on the adhesion of BMSCs was verified by the Q-PCR assay and Western Blot assay.Fourth.the number of suspended cells reflects the change trend of hematopoietic cells after DBT treated.The cell cycle test can reflect the indirect effect of DBT on the cell cycle of hematopoietic cells.The effect of DBT on the formation of erythroid cells was estimated by using flow cytometry to analyze the proportion of early/middle/late erythroid cells in hematopoietic cells.Subsequently,the ability to differentiate erythroid cells from erythroid progenitor cells could be verified by CFU-E experiment.Result:Ferulic acid and fermononetin were used as traditional Chinese medicine markers for Radix Angelicae Sinensis and Radix Astragali,respectively.The chromatogram of Danggui Buxue Tang was achieved by High Performance Liquid Chromatography(HPLC).The elution time of ferulic acid and fermononetin was 25 min and 59 min,respectively,and the maximum absorption peak at 323.4 nm and 249.9 nm in the visible range,respectively.After comparison and analysis,the two substances also exist in the Danggui Buxue Tang.The similarity between the three times chromatographic fingerprint and the previous control map of the laboratory reached 97.4%and RSD was 1.22%,indicating that the three extractions of DBT components were basically consistent in general.The similarity between the chromatographic fingerprints obtained from different time points and their common atlas was more than 95%,RSD is 0.67%.These results showed that the samples can maintain good stability in the 24h.According to these experimental results,the quality control of Danggui Buxue Tang can be set up.On the basis of previous studies,the optimal density of bone marrow stromal cells was 3.2×10~6~4.8×10~6个/mL(1×10~6~1.5×10~6个/cm~2)in vitro.When the concentration of DBT was 0.5 mg/mL~16 mg/mL,the number of BMSCs can be increased and the cell culture time in vitro was prolonged,but the cell cycle was inhibited.8 mg/mL DBT significantly promoted the adhesion and migration of BMSCs cells.The results of fluorescent staining showed that BMSCs,treated with DBT,expressed more adherent protein,and the cytoskeleton changed obviously,and the cell extension was bigger,and Pseudopodia appeared obviously.High-throughput sequencing experiments have predicted that gene transcription in BMSCs is related to adhesion mechanism and has the strongest correlation with the Focal Adhesion signaling pathway.Q-PCR and WB experiments confirmed that DBT can promote the expression of Vinculin and cytoskeletal protein by affecting Focal adhesion signaling pathway,thereby affecting the adhesion and migration of BMSCs.After co-culture of DBT and bone marrow cells 36h,the number of suspended cells decreased,but there was no significant difference in the number of cells,and there was no obvious effect on the cell cycle.However,when DBT was treated with 30h,the number and proportion of erythroid cells in the early and middle stages increased significantly,and the number and proportion of late erythroid cells decreased significantly.The results showed that low concentration of DBT could promote the undifferentiated ability of erythroid progenitor cells.With the increase of concentration,the undifferentiated ability of erythroid progenitor cells was weakened,which promot ing the formation of erythroid cells.Conclusion:in the premise of ensuring the quality of the drug with the fingerprint of Chinese medicine,Danggui Buxue Tang has contributed to bone marrow stromal cells cultured in vitro,and promotes cell proliferation,adhesion and migration.The results showed that DBT can promote the expression of the bone marrow stromal cells through the Focal adhesion signaling pathway to induce the change of cytoskeleton protein.It is guessed that the decoction may indirectly promote the differentiation of hematopoietic progenitor cells into red blood cells by affecting the adhesion of bone marrow stromal cells.