Rainbow Trout (Oncorhynchus mykiss) Spleen-Derived Bioactive Compounds Suppress Pro-Inflammatory Gene Networks via NF-κB Pathway Modulation

Background: Natural products are key sources of anti-inflammatory agents, yet the potential of fish visceral extracts remains largely unexplored. This study evaluated the anti-inflammatory activity of a spleen extract from rainbow trout (Oncorhynchus mykiss). Methods: A crude spleen extract and its four solvent fractions were tested in LPS-stimulated RAW264.7 macrophages. Nitric oxide production and expression of iNOS, COX-2, and cytokines were assessed by qRT-PCR and Western blotting. The most active fraction, OSB (n-butanol layer), was further analyzed for its effects on NF-κB signaling, macrophage polarization, and ROS generation. Results: The crude spleen extract significantly reduced NO production and downregulated iNOS and COX-2 expression at both the transcriptional and translational levels. Among the four fractions, the OSB fraction exhibited the most potent and consistent anti-inflammatory effects. OSB markedly suppressed LPS-induced expression of iNOS, COX-2, and pro-inflammatory cytokines, while enhancing anti-inflammatory cytokines. Mechanistic analyses demonstrated that OSB inhibited NF-κB activation by preventing the nuclear translocation of the p65 subunit. Additionally, OSB attenuated LPS-induced ROS production and reduced the expression of M1 macrophage markers, indicating inhibition of M1 polarization. Conclusions: The OSB fraction from rainbow trout spleen exhibits potent anti-inflammatory activity by modulating the NF-κB pathway and suppressing M1 macrophage polarization, suggesting its potential as a natural therapeutic agent.