Molecular characterization, transcriptional profiling, and antioxidant activity assessment of nucleoredoxin (NXN) as a novel member of thioredoxin from red-lip mullet (Planiliza haematocheilus)

Nucleoredoxin (NXN) is a prominent oxidoreductase enzyme, classified under the thioredoxin family, and plays a pivotal role in regulating cellular redox homeostasis. Although the functional characterization of NXN has been extensively studied in mammals, its role in fish remains relatively unexplored. In this study, the NXN gene from Planiliza haematocheilus (PhNXN) was molecularly and functionally characterized using in silico tools, expression analyses, and in vitro assays. The predicted protein sequence of PhNXN contained 418 amino acids with an anticipated molecular mass of 47.53 kDa. It comprised a highly conserved ¹⁸⁸CPPC¹⁹¹ catalytic motif in the central NXN domain and two thioredoxin-like domains enriched with conserved Cys residues. PhNXN protein was primarily localized in the cytoplasm and nucleus of the cells. The spatial and temporal expression analyses of PhNXN mRNA showed the highest expression level in the brain under normal physiological conditions, while a significant modulation was detected in the blood and head kidney following immunostimulation with polyinosinic: polycytidylic acid, lipopolysaccharides, and Lactococcus garvieae. Recombinant PhNXN protein exhibited DPPH radical scavenging, thiol-dependent disulfide reduction, and cupric ion reduction activities. Additionally, PhNXN overexpression significantly suppressed oxidative stress-induced cell death, heavy metal cation-induced reactive oxygen species production, and viral hemorrhagic septicemia virus-induced cellular apoptosis in fish cells. Furthermore, PhNXN overexpression in RAW 264.7 cells demonstrated notable nitric oxide scavenging activity, M2-type polarization, and anti-inflammatory effect. Collectively, the overall findings of the study highlight the antioxidant and immunological functions of PhNXN in red-lip mullet.