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Myeloid-derived suppressor cells(MDSC)have been identified as a population of immature myeloid cells that suppress anti-tumor immunity.MDSC are increased in tumor-bearing hosts;thus,depletion of MDSC may enhance anti-tumor immunity.Astragalus flavonoids(TFA)is the extract of traditional chinese medicine Astragalus.The ability of TFA to modulate anticancer immunity has been recently extensively studied.However,the effect of TFA on MDSC has remained largely unexplored and whether TFA has Synergistic effect on breast cancer chemotherapeutic agent cyclophosphamide(CTX)are still unknown.The present study aimed to observe the Synergistic effect of Astragalus flavonoids(TFA)on breast cancer chemotherapeutic agent cyclophosphamide(CTX)and its regulation effect in immunologic function.Balb/C mice were injected with 4T1 mammary tumor cells to establish 4T1 breast cancer bearing mice model and then randomly divided into control group,model group,CTX group(80mg/Kg),CTX(80mg/Kg)combined with TFA(6mg/Kg)group and TFA group(6mg/Kg).Tumor sizes were measured and recorded every 3 days.After 3weeks of different treatments,tumor inhibition rates were measured;Spleen,bone marrow and blood were collected.Cell suspensions of these tissues were subsequently prepared for flow cytometry analysis.Subpopulation of the lymphocytes,marrow derived suppressor cells(MDSCs)in circulating blood were detected by Flow cytometry.Compared with model group,both CTX group and CTX-TFA group can significantly inhibit tumour growth with inhibition rate 55.61%(p<0.05)and 70.91%(p <0.01),respectively.However,compare to CTX group,CTX-TFA group showed a higher inhibition rate(p <0.05).Inhibitory effect of TFA on the growth of 4T1 breast cancer cells in vitro revealed that TFA has no inhibitory effect on 4T1 cells proliferation.Compared with CTX group,CTX-TFA group showed an increased percentage of CD3+,CD4+T and CD8+T lymphocytes(p <0.05 or p <0.01)in spleen and circulating blood,whereas the percentage of CD 19+cells remained unchanged.Besides,ATX-TFA group showed a decreased rate of regulatory T cells(Treg cells).In addition,CTX-TFA group can significantly decrease the percentage of MDSCs(CD11b+Gr1+)(p <0.05 or p <0.01).Further study found that CTX-TFA can both decrease CD11b+Ly6C+and CD11b+Ly6C-cells,which are two subsets of MDSC.Suppression assay for T cell proliferation revealed that CTX-TFA can relieve the inhibitory effect of MDSC-enriched BM cells on T cell proliferation,including CD4+T and CD8+T lymphocytes.Mechanistically,ATX-TFA increased the apoptosis of CD11b+Gr1+cells(almost MDSC)compared with that of CD11b+Gr1-cells.ROS in MDSC of CTX-TFA group were significantly increased when compared with CTX group,suggesting that TFA induced increase in MDSC apoptosis due to increased intracellular ROS might partially account for the observed depletion of MDSC.TFA could enhance the anti-tumour effect of CTX and the synergistic effect probably result of improving tumour immunosuppression via increasing the percentage of CD3+,CD4+T and CD8+T lymphocytes and downregulating the percentage of MDSCs.These findings suggest that elimination of MDSC using TFA may contribute to the overall anti-tumor properties,highlighting a novel property of TFA as potent MDSC-targeting agents,which may be used to enhance the efficacy of immunotherapeutic regimens.