Neuropeptide FF enhances antimicrobial defense by promoting intestinal barrier function in grass carp (Ctenopharyngodon idella).

The intestinal barrier serves as the first line of defense against enteric pathogens in fish. However, the neuroimmune regulatory mechanisms that govern its integrity, particularly those involving evolutionarily conserved neuropeptides, are largely unexplored in teleosts. Neuropeptide FF (NPFF), a member of the FMRFamide family, is known to modulate immune functions in mammals, yet its role in fish mucosal immunity remains poorly understood. In this study, we investigated the role of NPFF in the intestinal immunity of grass carp (Ctenopharyngodon idella). The NPFF gene encoded a 151-amino acid precursor protein with characteristic structural motifs shared across vertebrates. Tissue distribution analysis demonstrated high NPFF expression in the brain and moderate levels in immune-relevant organs, including the intestine. Bacterial challenge with Aeromonas hydrophila significantly upregulated NPFF expression in the intestine, spleen, and kidney, suggesting its involvement in immune responses. In vitro, recombinant grass carp NPFF promoted epithelial cell proliferation and upregulated genes associated with cell survival and proliferation. In vivo, grass carp NPFF administration enhanced intestinal barrier integrity by upregulating tight junction genes (ZO-1, Occludin, Claudin-1) and promoting epithelial cell proliferation. Furthermore, NPFF treatment increased the expression of immunoregulatory factors (AhR, IL-22), the mucosal barrier component MUC2 and antimicrobial peptides (LEAP-2, Lyz1, Hepcidin-1), which collectively led to a reduced bacterial load and improved survival rates. These findings demonstrate that NPFF plays a critical role in fortifying the intestinal mucosal barrier and enhancing antibacterial immunity in grass carp, highlighting its potential as a therapeutic target for aquaculture disease management.