Nonpolysaccharide fraction of Lonicerae japonicae Flos attenuates cyclophosphamide-induced immunosuppression associated with modulation of the Keap1/Nrf2/HO-1/GPX4 signalling pathway

Background Cyclophosphamide (CTX)-induced immunosuppression is closely associated with oxidative stress and ferroptosis, but effective therapeutic interventions remain limited. This study investigated the protective effects of the non-polysaccharide fraction of Lonicerae japonicae Flos (LJFE) against CTX-induced immunosuppression and explored its underlying molecular mechanism. Methods LJFE was prepared by water extraction followed by ethanol precipitation, and its chemical profile was characterized using UPLC-MS/MS. Mice with CTX-induced immunosuppression were administered LJFE at doses of 100, 200, and 400 mg/kg for 10 consecutive days. Immune organ indices, serum IgE and IgM levels, oxidative stress markers (SOD, GSH, MDA), and the TBX21/GATA3 ratio were measured. The Keap1/Nrf2/HO-1/GPX4 signalling pathway was investigated via Western blotting and immunofluorescence. Serum metabolomics and molecular docking were applied to screen key differential metabolites and predict binding interactions between characteristic components and target proteins. LPS-stimulated RAW264.7 macrophages were used to verify pathway activation. Results Seventeen chemical constituents were identified in LJFE. In vivo , LJFE markedly restored thymus and spleen indices, increased serum IgE and IgM levels by approximately 45% and 35%, respectively, elevated SOD and GSH activities, reduced MDA accumulation, and normalized the TBX21/GATA3 ratio. Mechanistically, LJFE downregulated Keap1 and upregulated Nrf2, HO-1, and GPX4 expression. Metabolomics analysis identified hydroferulic acid as a key differential biomarker involved in antioxidative regulation. Molecular docking suggested that characteristic components of LJFE exhibit favorable binding affinity toward Keap1 and Nrf2. In vitro , LJFE dose-dependently activated the Keap1/Nrf2/HO-1 signalling axis and suppressed excessive NO release in LPS-challenged macrophages. Conclusion LJFE ameliorates CTX-induced immunosuppression by modulating the Keap1/Nrf2/HO-1/GPX4 pathway, thereby alleviating oxidative stress and ferroptosis. These findings support the potential of LJFE for further development as an immune-enhancing functional food ingredient.