Research On Effect Of Osmotic Stress On Kidney-derived Cell Lines From Spotted Scat(Scatophagus Argus) And Hepcidin, A Novel Antimicrobial Peptide

The spotted scat, Scatophagus argus, is a euryhaline teleost. The strong capacity of different salinities makes spotted scat a great animal model to study the underlying osmoregulatory mechanisms, which will supply basic information for euryhaline teleost osmoregulation, serve for a better understanding of fish physiology and developmental biology, and provide theoretical foundation for fish salinity domestication so as to better instruct the aqualculture of Scatophagus argus.1. To better understand the osmoregulatory mechanisms and more accurately analysis the characteristics of adaptive responses to the osmotic pressure in fish, we developed a S. argus kidney-derived cell line(SK) and subcultured the cell for more than 40 passages, which enable a further analysis of the osmoregulatory mechanisms of the adaptive responses to the osmotic pressure in the cellular level. The kidney-derived cells are mostly fibroblast-like, with a normal diploid karyotype(2n=48). A low-osmolarity-adapted SK cell line(SK-la) grow well in a hypotonic medium(150 mOsm). To better analysis the effects of different osmotic pressure(150, 300 and 450 mOsm) on cell morphology, cell proliferation, cell volume changes and cell damage in SK cells(kidney-derived cell line from euryhaline teleost spotted scat), SK-la cells and CIK cells(kidney-derived cell line from freshwater teleost grass carp), we used microscope, flow cytometry and Na~+/K~+-ATPase activity assay. The cell morphology of SK cells became smaller and the growth rate was increased in response to hypotonic medium(150 mOsm). The cell morphology was unconspicuous changed in response to hypertonic medium(450 mOsm), but was slightly larger compared with the isotonic culture conditions. The growth of SK-la cells in hypertonic medium(300 mOsm) is good, but depressed in relatively more hypertonic medium(450 mOsm), and SK-la cells were more and more unhealthy with medium osmotic concentration increased. In contrast, CIK cells became unhealthy with anisotonic challenge, the growth rates in hypotonic medium(150 mOsm) and hypertonic medium(450 mOsm) were lower than that in the isotonic osmotic pressure(300 mOsm). The NKA activities of SK and CIK cells were assayed after exposure to anisotonic conditions, and rapid decreases were detected immediately except SK cells which were not affected in hypotonic medium(150 mOsm). Unlike SK, CIK cells, the NKA activity of SK-la cells in moderate hypertonic medium was increased first and then following a down-regulation. The experimental results showed that SK, SK-la cells had stronger osmoregulatory capacity than CIK cells in response to anisotonic stress, and provided a better understanding of osmosensing and osmoregulation mechanism in euryhaline fish kidney.2. Acute salinity decrease reduces the immunity of fish, and two SSH cDNA libraries generated from euryhaline teleost spotted scat were constructed, and the immune system was activated by acute decreased salinity. Hepcidin gene fragments were found from established transcriptome library of spotted scat. Hepcidins are small cysteine-rich antimicrobial peptides that play an important role in host immunity against pathogenic organisms. In this study, two hepcidins, SA-hepcidin1 and SA-hepcidin2, were cloned from spotted scat. SA-hepcidin1 belongs to the HAMP1 class, the length of open reading frame(ORF) is 273 bp, encoding 90 amino acids. SA-hepcidin2 belongs to the HAMP2 class, the length of open reading frame(ORF) is 267 bp, encoding 88 amino acids. Tissue localization was conducted by semi RT-PCR, the result revealed that SA-hepcidin1 was dominantly expressed in the spotted scat liver and brain, and also expressed in kidney, spleen, gill, eyes, notochord, intestine, heart,skin and muscle, while SA-hepcidin2 was only expressed in liver. In addition, mature SA-hepcidin peptides were synthesized to allow evaluation of their antimicrobial and antiviral functions in vitro. The synthetic SA-hepcidins had similar levels of antibacterial activity against Gram-positive and Gram-negative bacteria; however, the antibacterial activity of SA-hepcidin1 was stronger than that of SAhepcidin2. Epithelioma papulosum cyprini(EPC) and grass carp fin(GCF) cells were used to analysis the synthetic SA-hepcidins antiviral activities against the viral Siniperca chuatsi rhabdovirus(SCRV) and largemouth bass Micropterus salmoides reovirus(MsReV).The results suggest that SA-hepcidin2 has antiviral ability, but SA-hepcidin1 did not. The results of this study suggest that SA-hepcidins are important multifunctional proteins in the spotted scat immune system that are involved in resistance to various pathogens.