Immunoregulatory effects of astragaloside Ⅳ on inflammatory macrophage RAW264.7
Based on network pharmacology and in vitro experiments,this study was performed to explore the possible mechanism of astragaloside Ⅳ(AS-Ⅳ)in the prevention and treatment of pneumonia by regulating immune function in RAW264.7 macrophages.Pharm Mapper,SwissTargetPrediction and STITCH databases were used to predict the target sites of AS-Ⅳ,while Genecard and DisGeNET databases were used to retrieve the pneumonia targets,and the intersection with AS-Ⅳ targets was obtained.Then STRING platform and Cytoscape software were used to construct the protein interaction network of the intersection targets,and the core targets were selected according to the degree value.The DAVID database was used for GO functional annotation and KEGG pathway enrichment analysis,and the KEGG database was further used to predict the possible pathways of AS-Ⅳ intervention in pneumonia.For in vitro experiment,macrophage RAW264.7 of logarithmic phase were randomly divided into 3 groups:control group(C),model group(M)and the AS-Ⅳ group(AS-Ⅳ).Except for group C,the other two groups were stimulated with LPS,and the AS-Ⅳ group was further intervened by AS-Ⅳ.CCK8 method was used to determine the effect of AS-Ⅳ on the proliferation of RAW264.7 cells;ELISA was used to determine the secretion levels of NF-κB,IL-1β,MCP-1 and Arg-1;qPCR was used to detect the gene expression of TLR4 and its downstream pathway molecules HMGB1 and TLR4 in each group.Network pharmacological analysis predicted that AS-Ⅳ had 158 targets and 112 intersection targets with pneumonia,while the enrichment analysis of KEGG pathway obtained 126 pathways.In consider with literature,HMGB1,TLR4,TRIF,Myd88 and NF-κB were identified as the target of pathway to be verified.Further in vitro experiments confirmed that NF-κB,IL-1β and MCP-1 in AS-Ⅳ group were significantly decreased as compared with M group,while Arg-1 was significantly increased,the expression of HMGB1,TLR4,TRIF and Myd88 genes were significantly decreased as compared with M group.Taken together,AS-Ⅳ can regulate the inflammatory immune responses of RAW264.7 through HMGB1/TLR4 related signaling pathways,which provides experimental basis and data support for clinical pneumonia treatment.
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