白藜芦醇抑制嗜水气单胞菌毒力作用研究
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摘要
为探索白藜芦醇(Resveratrol, Res)在水产动物细菌病防控中的应用价值,实验以淡水养殖中重要的细菌病原嗜水气单胞菌(Aeromonas hydrophila)为研究对象,通过设置药物浓度梯度,检测其对嗜水气单胞菌生长、生物膜形成和溶血活性的抑制作用,和对毒力及群感调控系统相关基因表达的影响;同时通过人工感染异育银鲫(Carassius auratus gibelio)试验检测其对鱼体保护作用和对鱼体炎症相关因子基因表达的影响。结果显示:白藜芦醇对嗜水气单胞菌的最小抑菌浓度(MIC)>1024μg/mL;浓度低于64μg/mL时,对菌株生长影响不显著;浓度≥32μg/mL时,对病原菌株生物膜形成和溶血活性具有显著抑制作用(P<0.05),且随剂量增加而增强。荧光定量RT-PCR结果分析发现白藜芦醇能引起嗜水气单胞菌群感调控系统中luxR和luxS基因分别显著上调和下调表达;外膜蛋白基因omp表达显著下降。人工感染试验发现攻毒前两小时腹腔注射25、50和100 mg/kg白藜芦醇处理组的异育银鲫死亡率显著下降,鱼体炎症相关的肿瘤坏死因子(TNF-α)和Ⅱ型干扰素(IFN-γ)的mRNA表达量也显著下降。研究表明药用植物大黄、虎杖等所含白藜芦醇成分能有效抑制嗜水气单胞菌毒力,降低鱼体炎症反应的功效;腹腔注射25—100 mg/kg白藜芦醇对感染病原菌的异育银鲫有一定保护作用。
Resveratrol(Res) is an important active ingredient in Chinese herbal medicines such as rhubarb and polygonum cuspidatum with antibacterial, anti-inflammatory and antioxidant properties. To explore its application value in the prevention and treatment of bacterial diseases in aquatic animals, we explored the antibacterial activity of resveratrol against Aeromonas hydrophila, an important bacterial pathogen in freshwater aquaculture. The growth, biofilm formation, hemolysis inhibition, and virulence related gene expression of A. hydrophila under different resveratrol concentrations were detected. The in-vivo protection of resveratrol was confirmed by artificially infecting crucian carp with A. hydrophila. The results showed that the minimal inhibitory concentration(MIC) of resveratrol against A. hydrophila was above 1024 μg/mL, and the concentration less than 64 μg/mL did not affect the growth ratio of A. hydrophila. Resveratrol with more than 32 μg/mL significantly inhibited the biofilm formation and hemolytic activity of A. hydrophila(P<0.05) in a dose-dependent pattern. Resveratrol mediated heavy moisture of bacterium group sense of regulation and of QS system by inducing luxR and reucing luxS gene expression. The expression of omp was significantly down-regulated by resveratrol. Artificial infections showed that the mortality rate of allogynogenetic crucian carp infection in the 25—100 mg/kg resveratrol-treated group was significantly lower than that of the control group, and the expression of TNF-α and IFN-γ mRNA was significantly decreased. This study showed that resveratrol can effectively inhibit the virulence of A. hydrophila and reduce the inflammatory response at 2500 mg/kg on fish infected with A. hydrophila.
Resveratrol(Res) is an important active ingredient in Chinese herbal medicines such as rhubarb and polygonum cuspidatum with antibacterial, anti-inflammatory and antioxidant properties. To explore its application value in the prevention and treatment of bacterial diseases in aquatic animals, we explored the antibacterial activity of resveratrol against Aeromonas hydrophila, an important bacterial pathogen in freshwater aquaculture. The growth, biofilm formation, hemolysis inhibition, and virulence related gene expression of A. hydrophila under different resveratrol concentrations were detected. The in-vivo protection of resveratrol was confirmed by artificially infecting crucian carp with A. hydrophila. The results showed that the minimal inhibitory concentration(MIC) of resveratrol against A. hydrophila was above 1024 μg/mL, and the concentration less than 64 μg/mL did not affect the growth ratio of A. hydrophila. Resveratrol with more than 32 μg/mL significantly inhibited the biofilm formation and hemolytic activity of A. hydrophila(P<0.05) in a dose-dependent pattern. Resveratrol mediated heavy moisture of bacterium group sense of regulation and of QS system by inducing luxR and reucing luxS gene expression. The expression of omp was significantly down-regulated by resveratrol. Artificial infections showed that the mortality rate of allogynogenetic crucian carp infection in the 25—100 mg/kg resveratrol-treated group was significantly lower than that of the control group, and the expression of TNF-α and IFN-γ mRNA was significantly decreased. This study showed that resveratrol can effectively inhibit the virulence of A. hydrophila and reduce the inflammatory response at 2500 mg/kg on fish infected with A. hydrophila.
引文
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[10]Frémont L.Biological effects of resveratrol[J].Life Sciences,2000,66(8):663-673
[11]Yan Y N,Xia S L,Tian H Y,et al.Effects of resveratrol supplementation on growth performance,immunity,antioxidant capability and disease resistance of blunt snout bream fed high-fat diet[J].Acta Hydrobiologica Sinica,2017,41(1):155-164[闫亚楠,夏斯蕾,田红艳,等.白藜芦醇对高脂胁迫团头鲂抗氧化能力、非特异免疫机能和抗病力的影响.水生生物学报,2017,41(1):155-164]
[12]Waffoteguo P,Lee D,Cuendet M,et al.Two new stilbene dimer glucosides from grape(Vitis vinifera)cell cultures[J].Journal of Natural Products,2001,64(1):136-138
[13]Wang W B,Lai H C,Hsueh P R,et al.Inhibition of swarming and virulence factor expression in Proteus mirabilis by resveratrol[J].Journal of Medical Microbiology,2006,55(10):1313-1321
[14]Zhou J W,Chen T T,Tan X J,et al.Can resveratrol,a quorum sensing inhibitor,function as an aminoglycoside antibiotic-accelerant against Pseudomonas aeruginosa[J]?International Journal of Antimicrobial Agents,2018,52(1):35-41
[15]Augustine N,Goel A K,Sivakumar K C,et al.Resveratrol-A potential inhibitor of biofilm formation in Vibrio cholera[J].Phytomedicine,2014,21(3):286-289
[16]Gao L,Yuan Y K,LüZ R,et al.The immunomodulatory function of resveratrol[J].Journal of Xi’an Jiaotong University(Medical Sciences),2003,24(2):121-123[高路,袁育康,吕卓人,等.白藜芦醇的免疫调节作用.西安交通大学学报(医学版),2003,24(2):121-123]
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[18]Vasudevan P,Nair M K,Annamalai T,et al.Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation[J].Veterinary Microbiology,2003,92(1-2):179-185
[19]Mao Y,Niu S,Xu X,et al.The effect of an adding histidine on biological activity and stability of pc-pis from Pseudosciaena crocea[J].PLoS One,2013,8(12):e83268
[20]Bostanghadiri N,Pormohammad A,Chirani A S,et al.Comprehensive review on the antimicrobial potency of the plant polyphenol resveratrol[J].Biomedicine&Pharmacotherapy,2017,95:1588-1595
[21]He Z,Huang Z,Zhou W,et al.Anti-biofilm activities from resveratrol against fusobacterium nucleatum[J].Frontiers in Microbiology,2016,7:1065
[22]Jung C M,Heinze T M,Schnackenberg L K,et al.Interaction of dietary resveratrol with nimal-associated bacteria[J].FEMS Microbiology Letters,2009,297:266-273
[23]Zhao J,Zhang F R,Chui Y,et al.In vitro biosynthesis and activity detection of quorum sensing signal molecule AI-2 of Aeromonas hydrophila[J].Microbiology China,2015,42(10):1858-1865[赵晶,张福蓉,崔一,等.嗜水气单胞菌群体感应信号分子AI-2的细胞外生物合成及活性检测.微生物学通报,2015,42(10):1858-1865]
[24]Han X,Bai H,Liu L,et al.The luxS gene functions in the pathogenesis of avian pathogenic Escherichia coli[J].Microbial Pathogenesis,2013,55(2):21-27
[25]Wang N.Comparative proteomic analysis between biofilm and planktonic cells of Aeromonas hydrophila and characteristics of related oriteins[D].Thesis for Doctor of science.Nanjing Agricultural University,Nanjing.2012[王娜.嗜水气单胞菌浮游态和生物被膜状态比较蛋白质组学及相关蛋白特性分析.博士学位论文,南京农业大学,南京.2012]
[26]Kovacikova G,Lin W,Skorupski K.The LysR-type virulence activator AphB regulates the expression of genes in Vibrio cholerae in response to low pH and anaerobiosis[J].Journal of Bacteriology,2010,192(16):4181-4191
[27]Chui Y,Zhao J,Zhang F R,et al.Construction of luxSgene deletion mutant strain in Aeromonas hydrophila ATCC7966[J].Chinese Journal of Zoonoses,2016,32(5):462-468[崔一,赵晶,张福蓉,等.嗜水气单胞菌ATCC7966 luxS基因缺失株构建及特性研究.中国人兽共患病学报,2016,32(5):462-468]
[28]Smith D,Wang J H,Swatton J E,et al.Variations on a theme:diverse N-acyl homoserine lactone-mediated quorum sensing mechanisms in gram-negative bacteria[J].Science Progress,2006,89(3-4):167-211
[29]Swift S,Karlyshev AV,Durant EL,et al.Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida:identification of the LuxRI homologues AhyRI and AsaRI and their congnate signal molecules[J].Journal of Bacteriology,1997,179(17):5272-5281
[30]Zhang X J.Construction and characterization of LuxS deleted mutant strain in Aeromonas hydrophila J-1[D].Nanjing Agricultural University,Nanjing.2009[张小军.嗜水气单胞菌J-1株luxS基因缺失株的构建及特性分析.硕士学位论文,南京农业大学,南京.2009]
[31]Howard S P,Garland W J,Green M J,et al.Nucleotide sequence of the gene for the hole-forming toxin aerolysin of Aeromonas hydrophila[J].Journal of Bacteriology,1987,169(6):2869-2871
[32]Wong C Y,Heuzenroeder M W,Flower R L.Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model[J].Microbiology,1998,144(2):291-298
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