| In recent years, the development of aquaculture in China has been the first in the world.With the high density, high yield and the degree of intensification were increased, resulting infrequent outbreaks of disease, it caused huge economic losses to the aquaculture industry.According to statistics, those diseases caused more than ten billion yuan economic lossesevery year. At present, chemical drug prevention is still the primary means of prevention andcontrol method to aquatic animal diseases. Long-term, high-frequency and high-dose use offishery drugs will inevitably lead to increased pathogen resistance, drug residues andenvironmental pollution problem. In order to protect aquatic production and food andenvironmental safety, studies shows that the immunizing power of aquatic animals can beincreased through the use of immunostimulants to achieve the purpose of enhancing itstolerance to the pathogen, thereby reducing the use of chemical drugs, reducing the adversefactors caused the use of drugs. Therefore, use immunostimulants to prevent the outbreak ofdisease were considered to be an effective way. In my previous study, we found that thesecondary metabolites isolated from Alcaligenes faecalis showed the ability to improvenatural immunity indicators of grass carp, such as increase the phagocytic activity of whiteblood cells or enhance the serum bactericidal activity. In this study, we isolated some activesubstances from Alcaligenes faecalis, and we use Ctenopharyngodon idell kidney (CIK) toevaluted the immune enhancement efficacy of the active substances. This study also examinedthe changes in cell viability induced by the isolated compounds.1. Cultivate the Alcaligenes faecalis in large scale, treat the both with high-speedcentrifugation, methanol elution, adsorbing by macroporous resin D110,pressure reductionconcentration step by step and procured the extractum of metabolites. Apply the gel silicacolumn chromatography of chromatographic fractionation technique to isolate the extractumof Alcaligenes faecalis and obtain the active monomer A and B. Based on the physical andchemical properties and their spectral properties, compounds A and B were identified asphenylacetic acid and cyclic-(Pro-Gly) decapeptide, respectively.2. Phenylacetic acid and cyclic-(Pro-Gly) decapeptide was added to the CIK cell culture medium with the concentration of1μg/mL,10μg/mL and100μg/mL, respectively. Intreating process, the sample were acquired at2h,8h and24h, qRT-PCR was used toinvestigate their effects on gene expressions of MyD88and four cytokines (IL-1β, TNF-α,IL-8and typeⅠ-IFN) in CIK cells. The obtained results revealed that the expression ofMyD88, IL-1β, TNF-α and typeⅠ-IFN were significantly higher than blank control group.And on other hand, compared with the blank control group,two metabolites showed asignificant downward effect on the expression of IL-8.3. In this test, MTT assay was employed to measure changes in cell viability, to analyzewhether these compounds showed toxic to cells. From these results we can prove that theimmune-enhancing activity is indeed caused by the two metabolites. The results demonstratedthat in the case of concentrations under the100μg/mL, two of the compounds show no toxicto the CIK cells at72h post-treatment. Moreover, cyclo-(Gly-L-Pro)5shows the effect onincrease proliferation of the CIK cells.In summary, those two compounds have the ability to stimulate the expression of grasscarp immune-related genes, and two of them were showing no toxic to the CIK cells in ourstudy. There results demonstrated that the phenylacetic acid and cyclic-(Pro-Gly) decapeptideshowed the potential to develop into a new immunostimulants. |
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