<scp>FFAR4</scp> Functions as a <scp>DHA</scp> Receptor to Attenuate <scp>LPS</scp> ‐Induced Inflammation via the <scp>cAMP</scp> / <scp>IκBα</scp> Pathway in Large Yellow Croaker ( <scp> <i>Larimichthys crocea</i> </scp> )

Docosahexaenoic acid (DHA) exerts anti-inflammatory effects via free fatty acid receptor 4 (FFAR4) in mammals and is widely used in aquatic feeds for its anti-inflammatory properties. However, the role of FFAR4 in the anti-inflammatory effects of DHA in teleosts remains unclear. In this study, FFAR4 overexpression in vivo could significantly suppress LPS-induced expression of pro-inflammatory genes (il-6, il-1β, and tnf-α) in the head kidney of large yellow croaker. In contrast, under LPS stimulation, FFAR4 knockdown via exogenous dsRNA significantly aggravated the expression of these pro-inflammatory genes. Further in vitro experiments demonstrated that FFAR4-mediated anti-inflammatory effects were observed only with DHA co-treatment under LPS stimulation. This regulatory activity was completely abolished in the absence of DHA, indicating that co-administration of DHA is essential for FFAR4 function. Furthermore, DHA effectively induces receptor internalization in HEK293T cells stably expressing LcFFAR4. DHA exposure rapidly mobilized intracellular calcium ions (Ca²⁺), elevated cyclic adenosine monophosphate (cAMP) levels, and enhanced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Elevated cAMP levels subsequently triggered cAMP response element-binding protein (CREB) phosphorylation, which upregulated IκBα transcription and suppressed LPS-driven inflammatory cascades. In conclusion, this study revealed that FFAR4 mediates the anti-inflammatory effects of DHA through the cAMP-CREB-IκBα signaling pathway, providing novel molecular targets for nutrition-based anti-inflammatory strategies in aquaculture.