| Annual global aquaculture production has more than tripled within the span of15years,from16.8million tons in1990to52.9million tons in2005. Despite of the huge success, suchhigh production of aquaculture involves the intensive system of management practices, whichrenders the fish highly susceptible to different pathogens, facilitates transmissions ofinfectious pathogens and frequently induces outbreaks of emerging diseases. Application ofvarious immunostimulants to activate or boost the innate immune system has been widelyaccepted as a good strategy to improve health of cultured fish and fight epidemics.In our previous study, it has been demonstrated that the fermentation products ofthermophilic Anoxybacillus flavithermus SX-4, isolated from a sludge sample of Dongda hotspring (Shaanxi, China), was capable of increasing the non-specific immune parameters incarp (Cyprinus carpio) such as bactericidal activity and phagocytic activity by peripheralblood leucocytes. The present study isolated two purified compounds from the cell-freecultures of A. flavithermus SX-4, by column chromatography on silica gel and SephadexLH-20prior to recrystallization. Chemical structure of the isolated compounds wascharacterized by spectral analysis (nuclear magnetic resonance and mass spectrometry). Forthe purpose of evaluating the immunostimulatory activity of the two compounds, qRT-PCRwas used to investigate their effects on immune-related gene expression in grass carp(Ctenopharyngodon idella) kidney (CIK) cells. This study also examined the changes in cellviability induced by the isolated compounds and LPS. The resulsts of this study aresummarized as follows:1. A. flavithermus SX-4was inoculated in nutrient broth for7days on a reciprocal shakerwater bath at55℃. The combined culture broth (100L) was chromatographed onmacroporous resin DM130and eluted with methanol. The eluents were concentrated undervacuum by a rotary evaporator at65℃yielding92.0g of methanol extract. Part of methanolextract (70.0g) was put through a silica gel column (silica gel:100–200mesh) and elutedwith a solvent mixture of petroleum ether/ethyl acetate and ethyl acetate/methanol affording423fractions (500mL each). Fractions showing similar chromatograms (analyzed by TLC)were combined to yield8new fractions A–H. Fr. D and Fr. G were further separated and purified by column chromatography on silica gel and Sephadex LH-20prior torecrystallization, eventually leading to the isolation of two compounds (A and B). Based onthe physical and chemical properties and their spectral properties, compounds A and B wereidentified as p-hydroxyphenylacetylamide and cyclo-(Gly-L-Pro), respectively.2. In order to evaluate the immunostimulatory activity of the two isolated compounds,qRT-PCR was used to investigate their effects on gene expressions of MyD88and fourcytokines (IL-1β, TNF-α, IL-8and typeⅠ-IFN) in CIK cells. Expression profiling of theselected immune-related genes in cells stimulated with known immunostimulatory agent LPSwas also examined. The obtained results revealed that both isolated compounds, as did LPS,augmented the transcribing level of MyD88, an important adaptor molecule in toll-likereceptor (TLR) signaling pathway, as early as2h exposure. These compounds also inducedgene expression of cytokines in CIK cells such as IL-1β, TNF-α and typeⅠ-IFN.3. A MTT assay was employed to measure changes in cell viability, to analyze whetherthese compounds are toxic to the cells. The results demonstrated that in the case ofconcentrations remained below100μg/mL, none of the test compounds is toxic to the CIKcells at72h post-treatment. Moreover, cyclo-(Gly-L-Pro) and LPS could increase proliferationof the CIK cells.In summary, the present study demonstrates the immunostimulatory properties of the twometabolites (p-hydroxyphenylacetylamide and cyclo-(Gly-L-Pro)) from thermophilic A.flavithermus SX-4in CIK cells, and highlights the potential of using both metabolites asimmunostimulants in fish aquaculture. |
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