Expression profiling and functional role of cyclooxygenase-2 in the immune and inflammatory responses of red-spotted grouper (Epinephelus akaara)

Cyclooxygenase-2 (Cox-2) is a well-studied enzyme and a significant medicinal target associated with various inflammatory disorders. However, its role in pathogen-induced inflammatory responses in fish remains poorly understood. This study characterized the structural and functional properties of a Cox-2 homolog from red-spotted grouper (Epinephelus akaara) (EaCox-2). The three-dimensional structure of EaCox-2 revealed a homodimer with two functional domains: a catalytic domain with two active sites and a membrane-binding domain. EaCox-2 transcripts were ubiquitously expressed in all tested tissues of E. akaara, with the highest expression in the gills, followed by the spleen. Immune stimulation with polyinosinic:polycytidylic acid (poly I:C), lipopolysaccharides (LPS), and nervous necrosis virus (NNV) led to significant upregulation in EaCox-2 transcripts 12 and 24 h post-injection in both gill and spleen tissues. EaCox-2 overexpression in murine macrophages triggered a pro-inflammatory response characterized by M1 macrophage polarization, upregulation of pro-inflammatory mediators such as TNF-α, IL-1β, and IL-6, and iNOS enzyme, enhanced production of reactive nitric oxide (NO), and mitochondrial depolarization. These findings highlight the crucial role of EaCox-2 in regulating immune and inflammatory responses in E. akaara, providing valuable insights into the molecular mechanisms underlying teleost immunity.