Systems pharmacology reveals the mechanism of Astragaloside IV in improving immune activity on cyclophosphamide-induced immunosuppressed mice

Ethnopharmacological relevance: Myelosuppression, also known as bone marrow suppression (BMS), is a pathological phenomenon of the decrease in the production of blood cells and further lead to immune homeostasis disorder. Astragalus mongholicus Bunge (AM, checked with The World Flora Online, http://www.worldfloraonline.org, updated on January 30, 2023) is a traditional Chinese medicine with efficacy of tonifying Qi and strengthening body immunity in thousands of years of clinical practice in China. Astragaloside IV (AS-IV) is a major active ingredient of AM, which plays an important role in regulating immune system through different ways. Aim of the study: This study was aimed to investigate the protective effect and mechanism of AS-IV on macrophages in vitro and cyclophosphamide (CTX)-induced immunosuppressive mice in vivo, and to provide experimental basis for the prevention and treatment of AS-IV in myelosuppression. Materials and methods: Based on network pharmacology and molecular docking technology, the core targets and signaling pathways of saponins of AM against myelosuppression were screened. And then, the immunoregulatory effect of AS-IV on RAW264.7 cells was investigated by cellular immune activity and cellular secretion analysis in vitro. In this way, the effects of AS-IV on the main potential targets of HIF-1 alpha/NF-kappa B signaling pathway were analyzed by qRT-PCR and Western blot methods. Furthermore, comprehensive analysis of the effects of AS-IV against CTX-induced mice were conducted on the basis of immune organs indices analysis, histopathological analysis, hematological analysis, natural killer cell activity analysis and spleen lymphocyte transformation activity analysis. In order to further verify the relationship between active ingredients and action targets, drug inhibitor experiments were finally conducted. Results: AS-IV, as a potential anti-myelosuppressive compound, was screened by systematic pharmacological methods to act on target genes including HIF1 alpha and RELA together with the HIF-1 alpha/NF-kappa B signaling pathway. Further studies by molecular docking technology showed that AS-IV had good binding activity with HIF1 alpha, RELA, TNF, IL6, IL1B and other core targets. Besides, cellular and animal experiments validation results showed that AS-IV could enhance the migration and phagocytosis of RAW264.7 cells, and protect the immune organs such as spleen and thymus together with bone tissues from damage. By this means, immune cell function including spleen natural killer cell and lymphocyte transformation activity were also enhanced. In addition, white blood cells, red blood cells, hemoglobin, platelets and bone marrow cells were also significantly improved in the suppressed bone marrow microenvironment (BMM). In kinetic experiments, the secretion of cytokines such as TNF-alpha, IL-6 and IL-1 beta were increased, and IL-10, TGF-beta 1 were decreased. The key regulatory proteins such as HIF-1 alpha, NF-kappa B, PHD3 in HIF-1 alpha/NF-kappa B signaling pathway were also regulated in the results of upregulated expression of HIF-1 alpha, p-NF-kappa B p65 and PHD3 at the protein or mRNA level. Finally, the inhibition experiment results suggested that AS-IV could significantly improve protein response in immunity and inflammation such as HIF-1 alpha, NF-kappa B and PHD3. Conclusion: AS-IV could significantly relieve CTX-induced immunosuppressive and might improve the immune activity of macrophages by activating HIF-1 alpha/NF-kappa B signaling pathway, and provide a reliable basis for the clinical application of AS-IV as a potentially valuable regulator of BMM.