Immune-Enhancement Effects of 6-Methoxykaempferol on Cyclophosphamide-Induced Immunosuppression via Improving Antioxidant Enzyme Expression, NF-κB and MAPK Signaling, and Modulating Gut Microbiome.

The immune system maintains homeostasis through coordinated innate and adaptive responses, and its imbalance increases disease susceptibility. The immunomodulatory effects of 6-methoxykaempferol (6MK), a methoxylated flavonoid found in sweet cherries, were studied in a mouse model of cyclophosphamide (CPA)-induced immunosuppression. The expression of key signaling proteins in the NF-κB and MAPK pathways was studied to explore the underlying molecular mechanisms. The Toll-like receptor-4/myeloid differentiation factor-2 receptor complex (TLR4/MD2), which can stimulate the immune response by activating these pathways, was used to study possible interactions with 6MK using docking analysis. 6MK administration significantly restored immune organ integrity (spleen up to 15.1% and thymus up to 16.8%), enhanced NK cell function (up to 43.8%), promoted T (up to 24.5%) and B cell proliferation (up to 26.4%), increased pro- and anti-inflammatory cytokine (IL-1β, IL-6, TNF-α, IL-4, IL-10, and TGF-β) levels, and elevated NO (up to 25.6%) and immunoglobulin (IgG, IgA, and IgM) concentrations. Additionally, 6MK upregulated antioxidant enzymes (CAT, HO-1, and SOD) and reactivated suppressed NF-κB and MAPK pathways. The docking-supported hypothesis, based on putative interactions and the estimated free energy of binding, suggests that 6MK possesses agonistic potential for the TLR4/MD2. Changes in the gut microbiome due to 6MK treatment, such as an increase in alpha diversity, abundance of Dorea longicatena, and the upregulation of formaldehyde-consuming pathways, may also contribute to immune enhancement. These findings show that 6MK may alleviate immunosuppression, suggesting its potential for future studies targeting immune-related diseases and conditions.