Hepcidin from Onychostoma macrolepis: Response to Aeromonas hydrophila infection by down-regulating expression of inflammatory factor genes and regulating iron homeostasis potentially.

ABSTRACT

Hepcidin, as an antimicrobial peptide, is associated with innate immunity and is considered a potential antibiotic substitute. In the present study, the hepcidin gene from the cavefish - Onychostoma macrolepis was identified and analyzed. The recombinant hepcidin protein (rOmhepc) was obtained by prokaryotic expression, evaluating the inhibitory effect of 5 pathogenic bacteria in vitro. Sixty O. macrolepis injected with 100 µL A. hydrophila (1.5 × 108 CFU/mL) were randomly divided into the therapeutic group and infection group, and therapeutic group was injected with 100 µL rOmhepc (100 µg/mL) at 6 and 18 h. The survival rates of O. macrolepis and bacterial load in liver were measured at 24 h. The liver tissues were collected at 0, 6, 12, and 24 h after A. hydrophila injection for investigating expression levels of immune-related, inflammatory factor genes and FPN1 gene. The results demonstrated that the hepcidin CDS contained 279 bp and encoded 93 aa. Hepcidin protein has a hydrophobic surface formed by multiple hydrophobic residues (CCGCCYC), and the theoretical pI was 7.53. Omhepc gene was expressed at varying levels in tested tissues, with the liver showing the highest expression, followed by the spleen. The expression of hepcidin gene following A. hydrophila infection was up-regulated and then down-regulated in liver, and the highest expression level was found at 12 h with a 10.93-fold. The rOmhepc remarkably inhibited the growth of A. hydrophila, Staphylococcus aureus, and Streptococcus agalactiae, with inhibition rates reaching 69.67 %, 42.97 %, and 65.74 % at 100 µg/mL. The mortality rates of O. macrolepis and bacterial load in liver were significantly decreased in the therapeutic group than that of infection group (p < 0.05). After the rOmhepc therapeutic, interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were significantly down-regulated with 14.4-fold and 106.07-fold at 24 h. Furthermore, the expression of immune-related genes (C3, TNF-α, IFN-γ) and Ferroportin gene (FPN1) significantly decreased (p < 0.05). The integrated analyses indicated that the rOmhepc could significantly inhibit the growth of A. hydrophila both in vitro and in vivo, attenuating the over-expression of inflammatory factor, FPN1 and immune-related genes.