酿酒酵母细胞壁可通过TLR2受体调控绵羊瘤胃上皮细胞表达SBD-1
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摘要
旨在研究酿酒酵母细胞壁对体外培养绵羊瘤胃上皮细胞β-防御素-1(sheep beta-defensin-1,SBD-1)表达的影响及其调控途径,为揭示酿酒酵母细胞壁对机体免疫的调控机制提供理论依据。本研究将不同质量浓度的酿酒酵母细胞壁提取物(0,25,50,100,200,400mg/L)作用于绵羊瘤胃上皮细胞不同时间(0,2,4,8,12,24h)后,检测上皮细胞SBD-1和Toll样受体-2(TLR2)mRNA表达水平,并进一步通过TLR2阻断试验来证实TLR2是否介导酵母细胞壁促进SBD-1表达。结果表明,不同浓度的酵母细胞壁刺激瘤胃上皮细胞时,随着细胞壁浓度的增加SBD-1mRNA表达量呈先升高后降低的趋势,且酿酒酵母细胞壁质量浓度为200mg/L刺激12h时,SBD-1 mRNA表达量达到峰值,与对照组和其他时间组相比差异显著(P<0.05);用质量浓度为200mg/L的酿酒酵母细胞壁刺激绵羊瘤胃上皮细胞不同时间,同样在12h时,TLR2的mRNA表达量达到最大;阻断试验表明TLR2可介导SBD-1的表达。本研究结果表明酿酒酵母细胞壁可促进绵羊瘤胃上皮细胞SBD-1的表达,且质量浓度200mg/L时,刺激瘤胃上皮细胞12h时SBD-1的表达量达到最高,而TLR2参与该调控过程。
The aim of this study was to explore the effect of cell wall of Saccharomyces cerevisiae(S.cerevisiae CW)on induction of sheep beta defensin-1(SBD-1)and correlated signal pathway cultured sheep ruminal epithelial cells(SOECs)and to provide a theoretical basis for revealing the regulatory mechanism of Sc.erevisiae CW in expression of SBD-1.In this study,SOECs were cultured,then exposed to Sc.erevisiae CW for 24 hours(0-24 h),with a range of concentrations(0-400 mg/L).RT-qPCR was performed to detect SBD-1 and TLR2 mRNA.Then,blocking TLR2,and detecting expression SBD-1 through TLR2 pathway.RT-qPCR results showed that the expression of SBD-1 mRNA increased at first and then decreased,with the increased concentrations of S.cerevisiae CW stimulating SOECs.The expression of SBD-1 mRNA reached a peak with the concentration of 200 mg/L at 12 h,and there were significant differences compared to the other groups(P<0.05).TLR2 mRNA also reached a peak,at the same condition and time point.And,block test further affirmed that Sc.erevisiae CW could increase SBD-1 expression in SOECs through TLR2 pathway.The results of this study indicated that S.cerevisiae CW could improve the expression of SBD-1 in SOECs.And the level of SBD-1 expression was the highest when the ruminal epithelial cells were stimulated with the cell wall concentration of 200 mg/L for 12 h,and TLR2 took part in the modulation of SBD-1 expression.
The aim of this study was to explore the effect of cell wall of Saccharomyces cerevisiae(S.cerevisiae CW)on induction of sheep beta defensin-1(SBD-1)and correlated signal pathway cultured sheep ruminal epithelial cells(SOECs)and to provide a theoretical basis for revealing the regulatory mechanism of Sc.erevisiae CW in expression of SBD-1.In this study,SOECs were cultured,then exposed to Sc.erevisiae CW for 24 hours(0-24 h),with a range of concentrations(0-400 mg/L).RT-qPCR was performed to detect SBD-1 and TLR2 mRNA.Then,blocking TLR2,and detecting expression SBD-1 through TLR2 pathway.RT-qPCR results showed that the expression of SBD-1 mRNA increased at first and then decreased,with the increased concentrations of S.cerevisiae CW stimulating SOECs.The expression of SBD-1 mRNA reached a peak with the concentration of 200 mg/L at 12 h,and there were significant differences compared to the other groups(P<0.05).TLR2 mRNA also reached a peak,at the same condition and time point.And,block test further affirmed that Sc.erevisiae CW could increase SBD-1 expression in SOECs through TLR2 pathway.The results of this study indicated that S.cerevisiae CW could improve the expression of SBD-1 in SOECs.And the level of SBD-1 expression was the highest when the ruminal epithelial cells were stimulated with the cell wall concentration of 200 mg/L for 12 h,and TLR2 took part in the modulation of SBD-1 expression.
引文
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[11]李砚,杨银凤.家畜体内防御素的多态性和表达[J].中国畜牧兽医,2013,40(3):160-168.
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[17]黎观红,洪智敏,贾永杰,等.鼠李糖乳酸杆菌LGA对鸡小肠上皮细胞β-防御素-9基因表达的影响[J].畜牧兽医学报,2012,43(4):634-641.
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[19]WANG Q,DZIARSKI R,KIRSCHNING C J,et al.Micrococci andpeptisoglycan activate TLR2→MyD88→IRAK→TRAF→NIK→IKK→NF-κB signal transduction pathway that induces transcription of interleukin-8[J].Infect Immun,2001,69(4):2270-2276.
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[21]范燕茹.乳杆菌对绵羊瘤胃上皮细胞中β-防御素表达的影响及其信号通路的初步研究[D].内蒙古呼和浩特:内蒙古农业大学,2016.
[22]WEHKAMP J,HARDER J,WEHKAMP K,et al.NF-κB and AP-1 mediated induction of human beta defensin-2in intestinal epithelial cells by Escherichia coli Nissle 1917:a novel effect of a probiotic bacterium[J].Infect Immun,2004,72(10):5750-5758.
[23]KLIS F M,BOORSMA A,DE GROOT P W.Cell wall construction in Saccharomyces cerevisiae[J].Yeast,2006,23:185-202.
[24]BROADWAY P R,CARROLL J A,BURDICKSANCHEZ N C.Live yeast and yeast cell wall supplements enhance immune function and performance in food-froducing livestock:a review[J].Microorganisms,2015,3(3):417-427.
[25]XIAO Z,TRINCADO C A,MURTAUGH M P.β-glucan enhancement of T cell ifnγresponse in swine[J].Vet Immunol Immunop,2004,102:315-320.
[26]孔维华,王京杭,彭正华等.β-1,3-葡聚糖抗实验性胃溃疡作用研究[J].山东大学学报(自然科学版),2001,50(1):107-112.
[27]TAO M A,YAN T U,ZHANG N F,et al.Effects of dietary yeastβ-glucan on nutrient digestibility and serum profiles in pre-ruminant Holstein calves[J].J Integr Agr,2015,14(4):749-757.
[28]李国辉,王金荣,苏兰利,等.饲用甘露寡糖在动物生产中应用研究[J].饲料博览,2017,30(11):20-23.
[29]许浩,申立泉,康坤,等.日粮添加酵母细胞壁对泌乳早期荷斯坦牛生产性能的影响[J].中国奶牛,2017,35(5):4-8.
[30]戈婷婷,瞿明仁,张晖.功能性寡糖对锦江黄牛瘤胃发酵及微生物生长效率的影响[J].动物营养学报,2012,24(3):557-562.
[31]SEMPLE F,DORIN J R.β-defensins:multifunctional modulators of infection,inflammation and more[J].JInnate Immun,2012(4):337-348.
[32]范燕茹,王佩,金鑫,等.乳杆菌诱导绵羊瘤胃上皮细胞内SBD-1的表达[J].中国兽医学报,2016,36(4):604-612.
[33]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.
[34]AKIRA S,TAKEDA K.Toll-like receptorsignalling[J].Nat Rev Immunol,2004(4):499-511.
[35]FITZGERALD K A,WHIRTER S M,FAIA K L,et al.IKK epsilon and TBK 1are essential components of the IRF3signaling pathway[J].Nat Immunol,2003(4):491-496.
[36]VORA P,YOUDIM A,THOMAS L S,et al.β-defensin-2expression is regulated by TLR signaling in intestinal epithelial cells[J].J Immunol,2004,173(9):5398-5405.
[2]GONCALVES B L,GONCALVES J L,ROSIM R E.Effect of different sources of Saccharomyces cerevisiae biomass on milk production,composition,and aflatoxin M-1excretion in milk from dairy cows fed aflatoxin B-1[J].J Dairy Sci,2017,100(7):5701-5708.
[3]SANTAS J,LAZARO E,CUNE J.Effect of a polysaccharide-rich hydrolysate fromSaccharomyces cerevisiae(LipiGo(R))in body weight loss:randomised,double-blind,placebo-controlled clinical trial in overweight and obese adults[J].J Sci Food Agr,2017,97(12):4250-4257.
[4]邵强,黄友解,韩月,等.酵母细胞壁的结构组成、生物学功能及在养殖业中的应用[J].浙江畜牧兽医,2017,42(1):13-16.
[5]中华人民共和国农业部公告第2045号[J].中国饲料,2014,25(1):1-4.
[6]SHEN Y B,PIAO X S,KIM S W,et al.Effects of yeast culture supplementation on growth performance,intestinal health,and immune response of nursery pigs[J].J Anim Ecol,2009,87(8):2614-2624.
[7]DIMITROGLOU A,MERRIFIELD D L,MOATE R,et al.Dietary mannan oligosaccharide supplementation modulates intestinal microbial ecology and improves gut morphology of rainbow trout,oncorhynchus mykiss(Walbaum)[J].J Anim Ecol,2009,87(10):3226-3234.
[8]常超,冯治敏,王琨,等.酿酒酵母细胞壁合生元对肉仔鸭肠道结构的影响[J].中国饲料,2014,25(14):13-15,24.
[9]MEADE K G,CORMICAN P,NARCIANDI F,et al.Bovineβ-defensin gene family:Opportunities to improve animal health?[J].Physiol Genomics,2014,46(1):17-28.
[10]ZHAO P,CAO G.Production of bioactive sheepβ-defensin-1in Pichia pastoris[J].J Ind Microbiol Biot,2012,39(1):11-17.
[11]李砚,杨银凤.家畜体内防御素的多态性和表达[J].中国畜牧兽医,2013,40(3):160-168.
[12]MONTELEONE G,CALASCIBETTA D,SCATUR-RO M,et al.Polymorphisms ofβ-defensin genes in Valle del Belice dairy sheep[J].Mol Biol Rep,2011,38(8):5405-5412.
[13]盛金良,陈创夫,杨霞.绵羊防御素(SBD1)mRNA在不同发育阶段的组织分布和定量分析[J].畜牧兽医学报,2009,40(1):20-25.
[14]唐博,曹贵方,吕东媛,等.蒙古绵羊雌性生殖道β-防御素(SBD-1)的序列分析及组织表达[J].中国兽医学报,2008,28(8):987-990.
[15]SCHLEE M,WEHKAMP J,ALENHOEFER A,et al.Induction of human beta-defensin 2by the probiotic Escherichia coli Nissle 1917is mediated through flagellin[J].Infect Immun,2007,75(5):2399-2407.
[16]刘佳明,丁卉,楼永良.乳杆菌细胞壁成分对小鼠阴道组织β-防御素-2诱导表达的影响[J].中国微生态学杂志,2011,23(4):306-309.
[17]黎观红,洪智敏,贾永杰,等.鼠李糖乳酸杆菌LGA对鸡小肠上皮细胞β-防御素-9基因表达的影响[J].畜牧兽医学报,2012,43(4):634-641.
[18]TAKEDA K,KAISHO T,AKIRA S.Toll-like receptors[J].Ann Rev Immunol,2003,21:335-376.
[19]WANG Q,DZIARSKI R,KIRSCHNING C J,et al.Micrococci andpeptisoglycan activate TLR2→MyD88→IRAK→TRAF→NIK→IKK→NF-κB signal transduction pathway that induces transcription of interleukin-8[J].Infect Immun,2001,69(4):2270-2276.
[20]成建国.酿酒酵母衰老过程中细胞壁组成及相关酶学性质研究[D].辽宁大连:大连工业大学,2012.
[21]范燕茹.乳杆菌对绵羊瘤胃上皮细胞中β-防御素表达的影响及其信号通路的初步研究[D].内蒙古呼和浩特:内蒙古农业大学,2016.
[22]WEHKAMP J,HARDER J,WEHKAMP K,et al.NF-κB and AP-1 mediated induction of human beta defensin-2in intestinal epithelial cells by Escherichia coli Nissle 1917:a novel effect of a probiotic bacterium[J].Infect Immun,2004,72(10):5750-5758.
[23]KLIS F M,BOORSMA A,DE GROOT P W.Cell wall construction in Saccharomyces cerevisiae[J].Yeast,2006,23:185-202.
[24]BROADWAY P R,CARROLL J A,BURDICKSANCHEZ N C.Live yeast and yeast cell wall supplements enhance immune function and performance in food-froducing livestock:a review[J].Microorganisms,2015,3(3):417-427.
[25]XIAO Z,TRINCADO C A,MURTAUGH M P.β-glucan enhancement of T cell ifnγresponse in swine[J].Vet Immunol Immunop,2004,102:315-320.
[26]孔维华,王京杭,彭正华等.β-1,3-葡聚糖抗实验性胃溃疡作用研究[J].山东大学学报(自然科学版),2001,50(1):107-112.
[27]TAO M A,YAN T U,ZHANG N F,et al.Effects of dietary yeastβ-glucan on nutrient digestibility and serum profiles in pre-ruminant Holstein calves[J].J Integr Agr,2015,14(4):749-757.
[28]李国辉,王金荣,苏兰利,等.饲用甘露寡糖在动物生产中应用研究[J].饲料博览,2017,30(11):20-23.
[29]许浩,申立泉,康坤,等.日粮添加酵母细胞壁对泌乳早期荷斯坦牛生产性能的影响[J].中国奶牛,2017,35(5):4-8.
[30]戈婷婷,瞿明仁,张晖.功能性寡糖对锦江黄牛瘤胃发酵及微生物生长效率的影响[J].动物营养学报,2012,24(3):557-562.
[31]SEMPLE F,DORIN J R.β-defensins:multifunctional modulators of infection,inflammation and more[J].JInnate Immun,2012(4):337-348.
[32]范燕茹,王佩,金鑫,等.乳杆菌诱导绵羊瘤胃上皮细胞内SBD-1的表达[J].中国兽医学报,2016,36(4):604-612.
[33]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.
[34]AKIRA S,TAKEDA K.Toll-like receptorsignalling[J].Nat Rev Immunol,2004(4):499-511.
[35]FITZGERALD K A,WHIRTER S M,FAIA K L,et al.IKK epsilon and TBK 1are essential components of the IRF3signaling pathway[J].Nat Immunol,2003(4):491-496.
[36]VORA P,YOUDIM A,THOMAS L S,et al.β-defensin-2expression is regulated by TLR signaling in intestinal epithelial cells[J].J Immunol,2004,173(9):5398-5405.
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