Study On Anti-myelosuppression Mechanism Of Astragaloside Ⅳ Regulating Immune Activity Of Macrophage Based On HIF-1α/NF-κB Signaling Pathway

Objective This study was aimed to investigate the regulatory effect of Astragaloside Ⅳ(AS-Ⅳ)on the immune activity of macrophage and the protective effect and mechanism of cyclophosphamide(CTX)-induced immunosuppressive mice,and to provide experimental basis for the application of AS-Ⅳ in the prevention and treatment of myelosuppression.Methods 1.TCMSP,ETCM,BATMAN-TCM and other databases were searched to obtain information of saponins of Astragalus membranaceus(AM).Swiss Target Prediction,Stitch and other databases were used to obtain and predict the potential targets of saponins of AM.Genecards and other databases were searched to obtain targets related to myelosuppression.STRING database,Venny 2.1.0 and Cytoscape 3.6.0 were used to construct PPI and"compound-target-disease" network of the common targets of saponins of AM against myelosuppression,so as to predict the core compounds and key targets of saponins of AM against myelosuppression.Metascape database was used for GO and KEGG pathway enrichment analysis on the common targets of saponins of AM against myelosuppression to predict the potential signaling pathways of saponins of AM against myelosuppression.2.The effect of AS-Ⅳ on the activity of macrophage RAW264.7 was detected by MTT assay,and the appropriate concentration of AS-Ⅳ was selected and the subsequent experiments were carried out.The macrophage RAW264.7 was randomly divided into negative control group,AS-Ⅳ group(1 μM,5 μM and 10 μM)and LPS group(1 μg/mL).The effect of AS-Ⅳ on the proliferation ability was detected by colony formation assay.The effect of AS-Ⅳ on the migration ability was detected by wound healing assay.The effect of AS-Ⅳon the adhesion ability was detected by crystal violet staining assay.The effect of AS-Ⅳ on the phagocytic ability was detected by neutral red assay.The effects of AS-Ⅳ on the secretion of NO and cytokines were detected by Griess and ELISA method respectively.The effects of AS-Ⅳ on ROS,MDA,SOD and GSH levels were detected by biochemical kits respectively.To detect the effects of AS-Ⅳ on the immune activity of macrophage RAW264.7.3.The effects of AS-Ⅳ on the expression of key genes and proteins of HIF-1α/NF-κB signaling pathway of macrophage RAW264.7 were detected by qRT-PCR and Western blot respectively.Western blot was used to detect the effect of inhibitors alone or in combination with AS-Ⅳ on the expression of key proteins.To study the mechanism of AS-Ⅳregulating the immune activity of macrophage RAW264.7.4.The C57BL/6J mice were randomly divided into 6 groups:normal control group(NC),model control group(MC),positive drug control group(PC,levamisole hydrochloride,40 mg/kg),AS-Ⅳ low-dose group(AS-Ⅳ-L,25 mg/kg),AS-Ⅳ medium-dose group(AS-Ⅳ-M,50 mg/kg)and AS-Ⅳ high-dose group(AS-Ⅳ-H,100 mg/kg).Except for the NC group,the mice in the other groups were intraperitoneally injected with CTX(80 mg/kg)on the 3rd,4th and 5th day to establish CTX-induced immunosuppressive model.During the administration,the general condition of mice was observed and the body weight was weighed every day.After administration,the blood was collected and all mice were sacrificed.The femur,spleen,thymus,liver,heart,lung and kidney was removed and weighed.The H&E staining was used to observe the pathological changes of the main organs and tissues of mice in each group.The hematological indices were detected by blood cell analyzer.The bone marrow cells were extracted and counted.The BMDM was induced by M-CSF and identified by flow cytometry.The phagocytic ability of BMDM was detected by neutral red method.The level of NO secreted by BMDM was detected by Griess method.The NK cell activity and lymphocyte transformation activity was detected by LDH and MTT method respectively.To study the immune function regulation and anti-myelosuppression of AS-Ⅳ in CTX-induced immunosuppressive mice in vivo.Results 1.A total of 335 common targets related to saponins of AM and myelosuppression.GO enrichment analysis showed that the common targets were mainly related to biological processes such as "transcription factor binding","regulation of small molecule metabolic process","transcription regulator complex" and so on.KEGG pathway enrichment analysis showed that the common targets were mainly related to "HIF-1 signaling pathway","NF-κB signaling pathway" and so on.PPI network analysis showed that key targets included HIF1A,IL1B,IL6,NOS3,RELA,TNF,VEGFA and so on,which were closely related to inflammation,immunity,hypoxia and HIF-1α/NF-κB signaling pathway.The results of"compound-target-disease" three-level network analysis showed that AS-Ⅳ was the core compound.The results of molecular docking showed that AS-Ⅳ had good binding activity with key targets such as HIF1A and RELA.2.AS-Ⅳ had no significant effect on the activity of macrophage RAW264.7 when the concentration was less than 25 μM.AS-Ⅳ could promote the colony formation,wound healing and adhesion of macrophage RAW264.7,enhance the ability of proliferation,migration,adhesion and phagocytosis.AS-Ⅳ could promote the secretion of NO,TNF-α,IL-6 and IL-1β,and inhibit the secretion of IL-10 and TGF-β1.AS-Ⅳ could increase the levels of ROS,MDA and GSH,and reduce the level of SOD.3.AS-Ⅳ could increase the mRNA expression level of HIF-1α,NF-κB p65,IKKβ,PHD3,TNF-α and IL-6,and reduce IKBa in macrophage RAW264.7,AS-Ⅳ could increase the protein expression levels of HIF-lα,p-NF-κB p65 and PHD3.AS-Ⅳ could reverse the decrease the protein expression of HIF-lα,p-NF-κB p65 and PHD3 induced by YC-1,PDTC and Roxadustat respectively.4.AS-Ⅳ could promote the increase of body weight and immune organ indices in CTX-induced immunosuppressive mice.AS-Ⅳ could repair the pathological changes of immune and major organs.AS-Ⅳ could increase the number of blood cells and bone marrow cells.AS-Ⅳ could enhance the NK cell activity and the lymphocyte transformation activity.AS-Ⅳ could enhance the phagocytosis of BMDM.AS-Ⅳ could promote the secretion of NO by BMDM and increase the levels of TNF-α,IL-6 and IL-1β in serum.Conclusion AS-Ⅳ could significantly alleviate CTX-induced myelosuppression symptoms,and might enhance the immune activity of macrophage by activating the HIF-1α/NF-κB signaling pathway,which provide a reliable experimental basis for the clinical application of AS-Ⅳ as a potential BMM regulator.