Ammopiptanthus nanus (M. Pop.) Cheng f. stem ethanolic extract ameliorates rheumatoid arthritis by inhibiting PI3K/AKT/NF-κB pathway-mediated macrophage infiltration.

Ethnopharmacological relevance: Ammopiptanthus nanus (M. Pop.) Cheng f. (A. nanus), a traditional Kirgiz medicinal plant, its stem has shown potential in treating rheumatoid arthritis (RA) in China, either through oral medication or by topical application directly to the affected joints, but its underlying mechanism of action remains unexplored.

Aim of the study: The purpose of this study is to elucidate pharmacological mechanism of A. nanus in ameliorating RA using a comprehensive approach that combines network pharmacology, molecular docking and experimental evaluations.

Materials and methods: Firstly, the major constituents of A. nanus stem ethanolic extract were identified and quantified by High-Performance Liquid Chromatography (HPLC). Disease target data from Gene Cards database was then used to define RA-associated targets. A protein-protein interaction (PPI) network was created via STRING database. The DAVID database powered gene ontology (GO) function and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to gain functional insights. In vitro, RAW264.7 cells were treated with A. nanus to investigate the roles of target proteins and pathways during lipopolysaccharide (LPS) - induced inflammation. Immunofluorescence assays were performed to assess the effects of A. nanus on macrophage infiltration. The key targets and signalling pathways were validated using enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR), molecular docking, immunohistochemical analysis, western blotting and immunofluorescence. Finally, the therapeutic potential of A. nanus in RA was evaluated in a carrageenan-induced rat model.

Results: Network analysis identified 31 potential targets of A. nanus associated with RA, including 10 hub targets. KEGG analysis highlighted the involvement of PI3K/AKT signaling pathway. In vivo experiments demonstrated that A. nanus treatment significantly protected against carrageenan-induced inflammatory paw tissue and attenuated macrophage infiltration. Both in vivo and in vitro experiments confirmed that A. nanus significantly downregulated the protein expression of COX-2, iNOS and IL-1β, and inhibited PI3K/AKT/NFκB pathway, which are closely linked to RA. Furthermore, molecular docking and cellular thermal shift assay revealed that licoflavanone showed a strong binding affinity with key targets.

Conclusion: In summary, this study provides the first evidence of the potent anti-inflammatory activity of A. nanus in experimental RA. The mechanism of action appears to involve inactivation of the PI3K/AKT/NF-κB pathway-mediated macrophage infiltration. These findings indicate that A. nanus has significant potential as a therapeutic potential agent for RA treatment and offer novel insights for future research and drug development in this field.