| 1.Background and Objective Chemotherapy is often accompanied by a side effect of leukopenia that affects its ongoing progression.Therefore,treatment of chemotherapy-induced leukopenia is a critical step in the sustainable and effective treatment of malignant tumors.Qi-JingSheng-Bai Granule(QJSB)is a traditional Chinese medicine formula for the treatment of leukopenia caused by radiotherapy and chemotherapy.It has been used in clinic for many years,but its active ingredients and potential mechanism of action have not yet been clarified.This study aims to confirm the efficacy of QJSB in the treatment of leukopenia,to reveal the pharmacodynamic basis of the formula,and to study its possible mechanism of action,thus providing scientific evidence and theoretical support for the development and application of QJSB.2.Method(1)Verifing the effects of QJSB: ICR mice were intraperitoneally injected with 80 mg/kg/d cyclophosphamide for 3 consecutive days to simulate a leukopenia model caused by chemotherapy.The mice were administered with low dose(1.5 g/kg/d)and high dose(3 g/kg/d)QJSB by gavage for 7 days,respectively,to verify the therapeutic effect of QJSB on leukopenia.(2)In vitro activity screening of the constituents absorbed into blood of QJSB: cell viability assay and colony formation assay in vitro were used to perform preliminary activity screening of the constituents absorbed into blood of QJSB,and to detect the influence of these constituents on the proliferation of murine primary bone marrow cells and murine bone marrow progenitor cell line 32 D cells.(3)Comparing the effects of the effective constituents formula with the original QJSB formula: the components that promoted cell proliferation in vitro were combined as a new formula(hereinafter referred to as the effective constituents formula)according to the content in the QJSB,and its efficacy on the mice leukopenia model was compared with the original formula,thus to further verify whether these ingredients were the effective constituents of QJSB.(4)Studying the mechanism of QJSB by network pharmacology: we used the techniques and methods of network pharmacology to search for the known and predicted targets of active components from the STITCH database,construct the PPI networks of these targets based on the STRING database,then perform KEGG pathway enrichment analysis based on the targets of these active components to determine the signal pathways that QJSB might affect,and verified the targets by Real-Time quantitive PCR,thus to reveal the mechanism of QJSB from the multi-levels of ‘chemical constituent,target,signal pathway'.3.Results(1)QJSB(1.5 g/kg/d)and(3 g/kg/d)significantly increased peripheral white blood cells and platelets,restored the imbalance of proportions of lymphocytes and eosinophils,increased the number of bone marrow nucleated cells and enhanced their cell viability,promoted the colony formation of bone marrow mononuclear cells,increased the spleen index,reversed the abnormal increase of serum IL-6 and G-CSF,and had a comprehensive therapeutic effect on leukopenia in mice.(2)The results of in vitro activity screening showed that among the 24 components absorbed into blood,14 constituents increased the cell viability of primary bone marrow nucleated cells and 32 D cells,and promoted the colony growth of primary bone marrow mononuclear cells.(3)The formula consisting of the 14 compounds also had a comprehensive therapeutic effect on leukopenia in mice,and there was no significant difference between its therapeutic effect and that of the original formula.(4)A total of 83 known and predicted targets of 14 compounds were found from the STITCH database.The results of the KEGG pathway enrichment analysis showed that multiple signaling pathways involved in cell development,immune response,biosynthesis and metabolism such as ‘PI3K-Akt signaling pathway',‘JAK-STAT signaling pathway',‘Th1 and Th2 cell differentiation',‘Th17 cell differentiation',‘T cell receptor signaling pathway',‘B cell receptor signaling pathway',‘Arginine and proline metabolism' and ‘Arginine biosynthesis' were significantly enriched.The results of RT-q PCR showed that QJSB and its effective constituents could regulate the m RNA expression of Jun,Bcl2l1,Nos3,Pik3 cg,Foxo3,Akt2 and Nfkbia genes both in vitro and in vivo.4 Conclusion This study confirmed that QJSB has a comprehensive therapeutic effect on cyclophosphamide-induced leukopenia in mice,and proved that 14 components absorbed into blood are the main effective constituents of the formula,and revealed that the formula might play a role through regulating multiple signaling pathways related to cell growth and development,immune response and biosynthesis and metabolism. |
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