酿酒酵母β-葡聚糖对绵羊瘤胃上皮细胞β-防御素-1表达的影响

doi:10.11843/j.issn.0366-6964.2018.11.013

摘要/Abstract

摘要：

为了探索β-葡聚糖对绵羊瘤胃上皮细胞（ruminal epithelial cells，RECs）β-防御素-1（sheep β-defensin-1，SBD-1）表达的影响。本研究首先建立绵羊RECs培养体系作为体外试验模型，用不同浓度（0、5、10、20、50和100 μg·mL^-1）的β-葡聚糖刺激RECs 8 h后，利用qPCR和ELISA方法检测RECs中SBD-1的表达变化，选出诱导SBD-1表达最高的β-葡聚糖浓度。然后用β-葡聚糖的最佳刺激浓度对RECs分别刺激0、2、4、8、12和24 h，同样利用qPCR和ELISA方法对RECs SBD-1的表达变化进行检测，从而筛选出诱导SBD-1表达最高的刺激时间。qPCR和ELISA结果显示，β-葡聚糖（5～100 μg·mL^-1）刺激RECs 8 h后，SBD-1 mRNA和蛋白的表达随着β-葡聚糖浓度的升高均呈现先升高后降低趋势，且当β-葡聚糖浓度为10 μg·mL^-1时SBD-1 mRNA和蛋白的表达量极显著高于对照组（P<0.01）。当用10 μg·mL^-1的β-葡聚糖刺激RECs 0～24 h后，qPCR结果显示，10 μg·mL^-1的β-葡聚糖刺激RECs 2 h时SBD-1 mRNA的表达量最高（P<0.01），之后呈下降趋势，而ELISA结果显示，在β-葡聚糖浓度为10 μg·mL^-1刺激RECs 4 h后SBD-1蛋白分泌水平达到最高（P<0.01）。MTT测定结果显示，100 μg·mL^-1β-葡聚糖会对绵羊RECs活力产生显著的影响（P<0.05）。本研究结果表明，β-葡聚糖能够提高绵羊瘤胃上皮细胞SBD-1的表达，且用浓度为10 μg·mL^-1的β-葡聚糖分别刺激RECs 2和4 h后SBD-1的mRNA和蛋白表达达到最高。

Abstract:

The aim of this study was to explore the effect of Saccharomyces cerevisiae β-glucan on the β-defensin-1(SBD-1) expression in cultured ruminal epithelial cells(RECs) of ovine. Firstly, the ovine RECs culture system was established as an in vitro experimental model, the RECs were stimulated with various concentrations (0, 5, 10, 20, 50 and 100 μg·mL^-1) of β-glucan for 8 h, and the expression level of SBD-1 mRNA and protein in RECs were detected by qPCR and ELISA to determine the concentration of β-glucan that induced the highest expression of SBD-1. Then, RECs were stimulated with the optimal stimulating concentration of β-glucan for 0, 2, 4, 8, 12 and 24 h, respectively and the expression of RECs SBD-1 were also detected by qPCR and ELISA, so as to determine the optimal stimulation time for β-glucan-induced SBD-1 expression. The qPCR and ELISA results showed that after β-glucan (5-100 μg·mL^-1) stimulated RECs for 8 h, the expression of SBD-1 mRNA and protein were increased and then decreased following the increase of concentrations, and when the concentration of β-glucan was 10 μg·mL^-1, the expression level of SBD-1 mRNA and protein were significantly higher than the control group (P<0.01). When RECs were stimulated with 10 μg·mL^-1 β-glucan for 0-24 h, qPCR results showed that SBD-1 mRNA expression was the highest at 2 h (P<0.01), and then downward; The results of ELISA test showed that SBD-1 protein secretion reached the highest level at 4 h (P<0.01).The results of MTT test showed that 100 μg·mL^-1 β-glucan significantly affect the sheep RECs viability (P<0.05). The results of this study indicated that β-glucan could improve the SBD-1 expression in RECs of ovine. And the level of SBD-1 mRNA and protein expression was the highest when the RECs were separately stimulated for 2 and 4 h with the 10 μg·mL^-1 β-glucan.

中图分类号:

S826.2

张曼, 金鑫, 王云鹤, 魏方, 温婧怡, 李政忆, 杨银凤. 酿酒酵母β-葡聚糖对绵羊瘤胃上皮细胞β-防御素-1表达的影响[J]. 畜牧兽医学报, 2018, 49(11): 2416-2424.

ZHANG Man, JIN Xin, WANG Yun-he, WEI Fang, WEN Jing-yi, LI Zheng-yi, YANG Yin-feng. Effects of Saccharomyces cerevisiae β-glucan on the Expression of SBD-1 in Ovine Ruminal Epithelial Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(11): 2416-2424.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2018.11.013

https://www.xmsyxb.com/CN/Y2018/V49/I11/2416

参考文献

[1] PESCHEL A,SAHL H G.The co-evolution of host cationic antimicrobial peptides and microbial resistance[J].Nat Rev Microbiol,2006,4(7):529-536.
[2] TAM J P,WANG S J,WONG K H,et al.Antimicrobial peptides from plants[J].Pharmaceuticals, 2015,8(4):711-757.
[3] GANZ T.Defensins:antimicrobial peptides of innate immunity[J].Nat Rev Immunol,2003,3(9):710-720.
[4] ZHU S Y,GAO B.Evolutionary origin of β-defensins[J].Dev Comp Immunol,2013,39(1-2):79-84.
[5] DONG J J,WU F,YE X,et al.β-defensin in Nile tilapia (Oreochromis niloticus):sequence,tissue expression,and anti-bacterial activity of synthetic peptides[J].Gene,2015,566(1):23-31.
[6] PELIZON A C,KANENO R,SOARES A M V C,et al.Immunomodulatory activities associated with beta-glucan derived from Saccharomyces cerevisiae[J].Physiol Res,2005,54(5):557-564.
[7] QI C J,CAI Y H,GUNN L,et al.Differential pathways regulating innate and adaptive antitumor immune responses by particulate and soluble yeast-derived β-glucans[J].Blood,2011,117(25):6825-6836.
[8] 徐智鹏,胡骏鹏,周小辉.酵母细胞壁多糖的作用及其成分检测方法[J].中国饲料,2015(11):40-42.
XU Z P,HU J P,ZHOU X H.The function of yeast cell wall polysaccharide and its detection methods[J].China Feed,2015(11):40-42. (in Chinese)
[9] SUZUKI I,TANAKA H,KINOSHITA A,et al.Effect of orally administered β-glucan on macrophage function in mice[J].Int J Immunopharmacol,1990,12(6):675-684.
[10] WALLER K P,COLDITZ I G.Effect of intramammary infusion of beta-1,3-glucan or interleukin-2 on leukocyte subpopulations in mammary glands of sheep[J].Am J Vet Res,1999,60(6):703-707.
[11] KOUGIAS P,WEI D,RICE P J,et al.Normal human fibroblasts express pattern recognition receptors for fungal (1→3)-β-_D-glucans[J].Infect Immun,2001,69(6):3933-3938.
[12] VAN DER MAREL M,ADAMEK M,GONZALEZ S F,et al.Molecular cloning and expression of two β-defensin and two mucin genes in common carp (Cyprinus carpio L.) and their up-regulation after β-glucan feeding[J].Fish Shellfish Immunol,2012,32(3):494-501.
[13] SCHMITT P,WACYK J,MORALES-LANGE B,et al.Immunomodulatory effect of cathelicidins in response to a β-glucan in intestinal epithelial cells from rainbow trout[J].Dev Comp Immunol,2015,51(1):160-169.
[14] 金鑫,张曼,范燕茹,等.酿酒酵母菌对绵羊瘤胃上皮细胞β-防御素-1(SBD-1)基因表达的影响[J].中国农业科学,2015,48(19):3910-3918.
JIN X,ZHANG M,FAN Y R,et al.Effects of saccharomyces cerevisiae on the expression of SBD-1 in cultured ruminal epithelial cells of sheep[J].Scientia Agricultura Sinica,2015,48(19):3910-3918. (in Chinese)
[15] THACKE P A.Alternatives to antibiotics as growth promoters for use in swine production:a review[J].J Anim Sci Biotechnol,2013,4(1):35.
[16] VAN DER FELS-KLERX H J,PUISTER-JANSEN L F,VAN ASSELT E D,et al.Farm factors associated with the use of antibiotics in pig production[J].J Anim Sci,2011,89(6):1922-1929.
[17] 王佩,范燕茹,金鑫,等.枯草芽孢杆菌对绵羊瘤胃上皮细胞β-防御素-1表达的影响[J].畜牧兽医学报,2015,46(5):760-767.
WANG P,FAN Y R,JIN X,et al.Effects of Bacillus subtilis on the expression of SBD-1 in cultured ruminal epithelial cells of sheep[J].Acta Veterinaria et Zootechnica Sinica,2015,46(5):760-767. (in Chinese)
[18] CUPERUS T,COORENS M,VAN DIJK A,et al.Avian host defense peptides[J].Dev Comp Immunol, 2013, 41(3):352-369.
[19] ZHANG G L,SUNKARA L T.Avian antimicrobial host defense peptides:from biology to therapeutic applications[J].Pharmaceuticals (Basel),2014,7(3):220-247.
[20] CAMPBELL Y,FANTACONE M L,GOMBART A F.Regulation of antimicrobial peptide gene expression by nutrients and by-products of microbial metabolism[J].Eur J Nutr,2012,51(8):899-907.
[21] ESTRADA A,YUN C H,VAN KESSEL A,et al.Immunomodulatory activities of oat β-glucan in vitro and in vivo[J].Microbiol Immunol,1997,41(12):991-998.
[22] VOLMAN J J,RAMAKERS J D,PLAT J.Dietary modulation of immune function by β-glucans[J].Physiol Behav,2008,94(2):276-284.
[23] SALEH M A D,AMORIM A B,GRECCO H A T,et al.Effects of β-(1→3,1→6)-_D-glucan and density of diets on the blood profiles of immunologically challenged weaned piglets[J].Int J Biol Macromol,2015,80:659-667.
[24] SHAO Y J,WANG Z,TIAN X Y,et al.Yeast β-_D-glucans induced antimicrobial peptide expressions against Salmonella infection in broiler chickens[J].Int J Biol Macromol,2016,85:573-584.
[25] CAMPOVERDE C,MILNE D J,ESTÉVEZ A,et al.Ontogeny and modulation after PAMPs stimulation of β-defensin,hepcidin,and piscidin antimicrobial peptides in meagre (Argyrosomus regius)[J].Fish Shellfish Immunol,2017,69:200-210.
[26] LEE I C,TOMITA S,KLEEREBEZEM M,et al.The quest for probiotic effector molecules-unraveling strain specificity at the molecular level[J].Pharmacol Res,2013,69(1):61-74.
[27] KIM K W,KANG S S,WOO S J,et al.Lipoteichoic acid of probiotic Lactobacillus plantarum attenuates Poly I:C-induced IL-8 production in porcine intestinal epithelial cells[J].Front Microbiol,2017,8:1827.
[28] CLUA P,KANMANI P,ZELAYA H,et al.Peptidoglycan from immunobiotic Lactobacillus rhamnosus improves resistance of infant mice to respiratory syncytial viral infection and secondary pneumococcal pneumonia[J]. Front Immunol,2017,8:948.
[29] LI Q,BAO F X,ZHI D F,et al.Lipopolysaccharide induces SBD-1 expression via the P38 MAPK signaling pathway in ovine oviduct epithelial cells[J].Lipids Health Dis,2016,15:127.