Active Ingredients and Potential Mechanism of Additive Sishen Decoction in Treating Rheumatoid Arthritis with Network Pharmacology and Molecular Dynamics Simulation and Experimental Verification

Background: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease in which macrophages produce cytokines that enhance inflammation and contribute to the destruction of cartilage and bone.Additive Sishen decoction (ASSD) is a widely used traditional Chinese medicine for the treatment of RA; however, its active ingredients and the mechanism of its therapeutic effects remain unclear.Methods: To predict the ingredients and key targets of ASSD, we constructed "drug-ingredient-target-disease" and protein-protein interaction networks.Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to explore the potential mechanism.The activity of the predicted key ingredients was verified in lipopolysaccharide-stimulated macrophages.The binding mode between the key ingredients and key targets was elucidated using molecular docking and molecular dynamics simulation.Results: In all, 75 ASSD active ingredients and 1258 RA targets were analyzed, of which kaempferol, luteolin, and quercetin were considered key components that mainly act through inflammation-related pathways, such as the PI3K-AKT, TNF, and IL-17 signaling pathways, to ameliorate RA.Transcriptome sequencing suggested that kaempferol-, luteolin-, and quercetin-mediated inhibition of glycolysis reduced the lipopolysaccharide-induced production of proinflammatory factors.In vitro experiments indicated that kaempferol, luteolin, and quercetin decreased Glut1 and LDHA expression by diminishing PI3K-AKT signaling to inhibit glycolysis.Molecular dynamic simulation revealed that kaempferol, luteolin, and quercetin stably occupied the hydrophobic pocket of PI3Kδ.Conclusion: Our results show that the PI3Kδ-mediated anti-inflammatory responses elicited by kaempferol, luteolin, and quercetin are crucial for the therapeutic efficacy of ASSD against RA.