Active ingredients and blood-entering sRNAs of Astragalus membranaceus attenuate inflammation through potential regulation of AKT-NF-κB signaling and sRNA-related targets

Objective Inflammation is a common pathological basis of numerous human diseases. Although Astragalus membranaceus exhibits pronounced anti-inflammatory activity, its underlying molecular mechanisms remain largely undefined. This study aimed to systematically dissect the anti-inflammatory basis of A. membranaceus —encompassing both bioactive small molecules and plant-derived small RNAs (sRNAs)—by integrating network pharmacology with in vitro and in vivo validation. Methods Active ingredients and candidate targets of A. membranaceus were identified by network pharmacology, and intersecting targets with inflammation were subjected to gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses. Hub targets were validated in lipopolysaccharide-stimulated RAW 264.7 macrophages by quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB), macrophage polarization assays, and enzyme-linked immunosorbent assay (ELISA). The regulatory effects of A. membranaceus -derived sRNAs on their predicted targets were assessed in parallel. Findings were further corroborated in a murine inflammation model using immunofluorescence, qPCR, WB, and ELISA. Results A total of 20 active ingredients and 204 targets shared between A. membranaceus and inflammation were identified. Enrichment analyses implicated the negative regulation of apoptosis and the interleukin-17A (IL-17) signaling pathway. In vitro , A. membranaceus downregulated serine/threonine (Ser/Thr)-protein kinase B alpha ( AKT1 ), reticuloendotheliosis viral oncogene homolog A ( RELA ), Jun proto-oncogene ( JUN ), and cyclin A2 ( CCNA2 ) at the transcriptional level, attenuated AKT1 and nuclear factor- κ B p65 (NF- κ B p65) phosphorylation, restored macrophage polarization, and reduced IL-6 sretion. A. membranaceus -derived sRNA1, sRNA2, and sRNA3 suppressed their predicted targets peroxisomal acyl-coenzyme A oxidase 3 ( ACOX3 ), ATP binding cassette subfamily C member 1 ( ABCC1 ), CD84 molecule ( CD84 ), and estrogen receptor 2 ( ESR2 ). These findings were confirmed in vivo : A. membranaceus downregulated AKT1 , RELA , JUN , and CCNA2 , reduced AKT1 phosphorylation, and lowered tumor necrosis factor- α (TNF- α ), IL-6, and IL-1 β levels, with consistent suppression of ACOX3 , ABCC1 , CD84 , and ESR2 . Conclusion A. membranaceus exerts its anti-inflammatory effects primarily through the AKT-NF- κ B signaling pathway, with an additional regulatory layer mediated by sRNAs targeting the ACOX3-ABCC1-CD84-ESR2 axis. By incorporating sRNAs into the network pharmacology framework, this study offers a more comprehensive understanding of the anti-inflammatory mechanisms of A. membranaceus .