Cyclovirobuxine D attenuates collagen-induced arthritis by inhibiting PI3K/AKT pathway and macrophage polarization.

Conventional therapeutic interventions for rheumatoid arthritis (RA) are frequently induced immunosuppressive side effects. The present study explores the therapeutic potential of Cyclovirobuxine D (CycD), a triterpenoid alkaloid isolated from Buxus microphylla, in a collagen-induced arthritis (CIA) mouse model or in vitro. The CIA model was treated with varying doses of CycD or methotrexate. The biomarkers of inflammatory factors related to macrophage polarization and oxidative stress in serum or synovial tissue was conducted. The RNA sequencing (RNA-seq) was employed to identify key post-regulatory genes in CycD treatment. The pathway was investigated using PI3K activators and inhibitors, both in vivo and vitro. Molecular docking, Network Pharmacology, Drug Affinity Responsive Target Stability (DARTS) and Cellular Thermal Shift Assay (CETSA) were performed to validate. The administration of CycD resulted in a substantial alleviation arthritis severity, joint pathology, and levels of biomarkers associated with macrophage polarization and oxidative stress in CIA mice. In contrast to methotrexate, CycD exhibited minimal adverse effects on the spleen and thymus. RNA-seq and immunofluorescent staining has showed that the biological function of CycD is predominantly characterized by its capacity to suppress the PI3K-AKT pathway in M1 macrophages. The activation of PI3K was found to counteract the therapeutic benefits of CycD in both CIA mice, RAW264.7 and THP-1 cells. Molecular docking, Network Pharmacology, DARTS and CETSA confirmed that CycD competes with PI3K for binding in M1-type macrophages. CycD alleviates collagen-induced arthritis by inhibiting the PI3K-AKT pathway in macrophages. GRAPHICAL ABSTRACT: [Figure: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13075-026-03754-5.