酿酒酵母菌及其灭活菌对绵羊瘤胃外植体β-防御素-1(SBD-1)表达的影响
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摘要
旨在探索益生性酿酒酵母菌及其灭活菌对绵羊瘤胃外植体内β-防御素-1(sheep beta-defensin-1,SBD-1)表达的影响。本研究建立了绵羊瘤胃外植体培养方法,将不同浓度(10~4、10~5、10~6、10~7、10~8、10~9 CFU·mL~(-1))的酿酒酵母菌及其灭活菌与外植体共培养24 h后,通过qPCR和ELISA方法检测瘤胃外植体内SBD-1 mRNA和蛋白的表达变化,以分别确定活菌及其灭活菌诱导SBD-1表达最高的菌液浓度;然后用该浓度的活菌及其灭活菌对瘤胃外植体进行不同时间(2、4、8、12、16、20、24 h)的刺激,同样用qPCR和ELISA方法检测瘤胃外植体内SBD-1 mRNA及蛋白的表达变化,从而筛选出活菌及其灭活菌诱导SBD-1表达的最佳时间。结果表明,益生性酿酒酵母菌及其灭活菌均能显著促进绵羊瘤胃外植体SBD-1的表达(P<0.05)。当酿酒酵母菌浓度为10~7 CFU·mL~(-1)、灭活菌浓度为10~8 CFU·mL~(-1)分别诱导绵羊瘤胃外植体16 h时,SBD-1表达量分别达到最大,且二者与对照相比均呈极显著差异(P<0.01)。在最佳诱导条件下对比二者的诱导效果,酿酒酵母菌活菌高于其灭活菌。因此,益生性酿酒酵母菌及其灭活菌均能促进绵羊瘤胃外植体内SBD-1的表达,且活菌浓度为10~7 CFU·mL~(-1)、灭活菌浓度为10~8 CFU·mL~(-1)分别诱导16 h时,绵羊瘤胃外植体内的SBD-1的表达量分别达到最大,并且益生性酿酒酵母菌的诱导效果高于其灭活菌。
The aim of this study was to explore the effects of probiotic Saccharomyces cerevisiae(S.c) and inactivated S.c on the expression of sheep-defensin-1(SBD-1) in rumen explants of sheep. After different concentrations(10~4, 10~5, 10~6, 10~7, 10~8, 10~9 CFU·mL~(-1)) of S.c and inactivated S.c were respectively co-cultured with explants for 24 h, the expression of SBD-1 mRNA and protein in rumen explants were detected by qPCR and ELISA to determine the optimal concentration for the induction of SBD-1 expression by S.c and inactivated S.c. Then, the sheep rumen explants were stimulated with the optimal concentration of S.c and inactivated S.c for 2, 4, 8, 12, 16, 20, 24 h, respectively, and the SBD-1 mRNA and protein were also detected by qPCR and ELISA, so as to screen out the optimal time for the induction of SBD-1 expression by S.c and inactivated S.c. The results showed that S.c and inactivated S.c could significantly promote the expression of SBD-1 in rumen explants(P<0.05). When the rumen explants of sheep were stimulated by the concentration of 10~7 CFU·mL~(-1) for S.c and 10~8 CFU·mL~(-1) for inactivated S.c for 16 h, respectively, the expression level of SBD-1 were the highest, and which were significantly different from the control(P<0.01). Comparing the induction effects between S.c and inactivated S.c under optimal induction conditions, the S.c were better. Therefore, S.c and inactivated S.c could promote the SBD-1 expression in rumen explants of sheep, and the expression of SBD-1 reached the maximum when the rumen explants of sheep were stimulated by the concentration of 10~7 CFU·mL~(-1) for S.c and 10~8 CFU·mL~(-1) for inactivated S.c for 16 h. The induction effect of S.c is better than that of inactivated S.c.
The aim of this study was to explore the effects of probiotic Saccharomyces cerevisiae(S.c) and inactivated S.c on the expression of sheep-defensin-1(SBD-1) in rumen explants of sheep. After different concentrations(10~4, 10~5, 10~6, 10~7, 10~8, 10~9 CFU·mL~(-1)) of S.c and inactivated S.c were respectively co-cultured with explants for 24 h, the expression of SBD-1 mRNA and protein in rumen explants were detected by qPCR and ELISA to determine the optimal concentration for the induction of SBD-1 expression by S.c and inactivated S.c. Then, the sheep rumen explants were stimulated with the optimal concentration of S.c and inactivated S.c for 2, 4, 8, 12, 16, 20, 24 h, respectively, and the SBD-1 mRNA and protein were also detected by qPCR and ELISA, so as to screen out the optimal time for the induction of SBD-1 expression by S.c and inactivated S.c. The results showed that S.c and inactivated S.c could significantly promote the expression of SBD-1 in rumen explants(P<0.05). When the rumen explants of sheep were stimulated by the concentration of 10~7 CFU·mL~(-1) for S.c and 10~8 CFU·mL~(-1) for inactivated S.c for 16 h, respectively, the expression level of SBD-1 were the highest, and which were significantly different from the control(P<0.01). Comparing the induction effects between S.c and inactivated S.c under optimal induction conditions, the S.c were better. Therefore, S.c and inactivated S.c could promote the SBD-1 expression in rumen explants of sheep, and the expression of SBD-1 reached the maximum when the rumen explants of sheep were stimulated by the concentration of 10~7 CFU·mL~(-1) for S.c and 10~8 CFU·mL~(-1) for inactivated S.c for 16 h. The induction effect of S.c is better than that of inactivated S.c.
引文
[1] MALACZEWSKA J,MILEWSKI S.Immunomodulating effect of Inter Yeast S on the non-specific and specific cellular and humoral immunity in lambs[J].Pol J Vet Sci,2010,13(1):163-170.
[2] PéREZ-RUCHEL A,REPETTO J L,CAJARUILLE C.Suitability of live yeast addition to alleviate the adverse effects due to the restriction of the time of access to feed in sheep fed only pasture[J].J Anim Physiol Anim Nutr (Berl),2013,97(6):1043-1050.
[3] GUEDES C M,GON?ALVES D,RODRIGUES M A M,et al.Effects of a Saccharomyces cerevisiae yeast on ruminal fermentation and fibre degradation of maize silages in cows[J].Anim Feed Sci Technol,2008,145(1-4):27-40.
[4] 陈惠娴,毛若雨,滕达,等.二硫键及其连接方式对防御素抗菌功能的影响研究进展[J].生物技术通报,2016,32(9):32-37.CHEN H X,MAO R Y,TENG D,et al.Review on effects of disulfide bond and its connection on antimicrobial activity in defensins[J]. Biotechnology Bulletin,2016,32(9):32-37.(in Chinese)
[5] ZHAO P W,CAO G F.Production of bioactive sheep β-defensin-1 in Pichia pastoris[J].J Ind Microbiol Biotechnol, 2012, 39(1): 11-17.
[6] XIAO Y J,HUGHES A L,ANDO J,et al.A genome-wide screen identifies a single β-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins[J].BMC Genomics,2004,5(1):56.
[7] MONTELEONE G,CALASCIBETTA D,SCATURROM,et al.Polymorphisms of β-defensin genes in Valle del Belice dairy sheep[J]. Mol Biol Rep,2011,38(8):5405-5412.
[8] 李砚,杨银凤.家畜体内防御素多态性和表达的研究进展[J].中国畜牧兽医,2013,40(3):160-168.LI Y,YANG Y F.Research progress of polymorphism and expression of defensins in vivo of livestock[J].China Animal Husbandry & Veterinary Medicine,2013,40(3):160-168.(in Chinese)
[9] 黎观红,洪智敏,贾永杰,等.抗菌肽的抗菌作用及其机制[J].动物营养学报,2011,23(4):546-555.LI G H,HONG Z M,JIA Y J,et al.Activities and mechanisms of action of antimicrobial peptides[J].Chinese Journal of Animal Nutrition, 2011,23(4):546-555.(in Chinese)
[10] SEMPLE F,DORIN J R.β-defensins:multifunctional modulators of infection,inflammation and more?[J].J Innate Immun, 2012, 4(4): 337-348.
[11] DASGUPTA T,NWEZE E I,YUE H,et al.Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53[J].Oncotarget,2016,7(19):27430-27444.
[12] CORLEIS B,LISANTI A C,K?RNER C,et al.Early type I Interferon response induces upregulation of human β-defensin 1 during acute HIV-1 infection[J].PLoS One,2017,12(3):e0173161.
[13] RYAN L K,DAI J H,YIN Z W,et al.Modulation of human β-defensin-1 (hBD-1) in plasmacytoid dendritic cells (PDC),monocytes,and epithelial cells by influenza virus,Herpes simplex virus,and Sendai virus and its possible role in innate immunity[J]. J Leukoc Biol,2011,90(2):343-356.
[14] 李砚.植物乳杆菌对绵羊瘤胃上皮细胞内SBD-1基因表达的影响[D].内蒙古:内蒙古农业大学,2013.LI Y.Effect of Lactobacillus plantarum on the expression of SBD-1 gene in rumen epithelial cells of sheep[D].Inner Mongolia:Inner Mongolia Agricultural University, 2013.(in Chinese)
[15] 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)
[16] JIN X,ZHANG M,ZHU X M,et al.Modulation of ovine SBD-1 expression by Saccharomyces cerevisiae in ovine ruminal epithelial cells[J].BMC Vet Res,2018,14(1):134.
[17] COSTA M O,HARDING J C,HILL J E.Development and evaluation of a porcine in vitro colon organ culture technique[J].in vitro Cell Dev Biol Anim,2016,52(9):942-952.
[18] SCHMITTGEN T D,ZAKRAJSEK B A.Effect of experimental treatment on housekeeping gene expression:validation by real-time,quantitative RT-PCR[J].J Biochem Biophysical Methods,2000,46(1-2):69-81.
[19] NAVA G M,ESCORCIA M,CASTA?EDA M P.Molecular diversity of the antimicrobial domain of Beta-defensin 3 and homologous peptides[J].Comp Funct Genomics,2009,2009:Article ID 983636.
[20] WANG S,ZENG X F,YANG Q,et al.Antimicrobial peptides as potential alternatives to antibiotics in food animal industry[J].Int J Mol Sci,2016,17(5):603.
[21] 田巧珍,金鑫,张曼,等.酿酒酵母细胞壁诱导体外培养绵羊瘤胃上皮细胞表达SBD-1的研究[J].畜牧兽医学报,2018,49(5):927-934.TIAN Q Z,JIN X,ZHANG M,et al.Effects of cell wall of Saccharomyces cerevisiae on the expression of SBD-1 in cultured ruminal epithelial cells of sheep[J].Acta Veterinaria et Zootechnica Sinica,2018,49(5):927-934.(in Chinese)
[22] SCHLEE M,HARDER J,K?TEN B,et al.Probiotic lactobacilli and VSL#3 induce enterocyte β-defensin 2[J].Clin Exp Immunol, 2008, 151(3):528-535.
[23] 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.LI G H,HONG Z M,JIA Y J,et al.Effect of Lactobacillus rhamnosus LGA on β-defensin 9 expression in cultured chicken small intestinal epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2012,43(4):634-641.(in Chinese)
[24] 范燕茹,王佩,金鑫,等.乳酸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.FAN Y R,WANG P,JIN X,et al.The expression of SBD-1 in the rumen epithelium cells of sheep induced by lactobacillus[J].Chinese Journal of Veterinary Science,2016,36(4):604-612.(in Chinese)
[25] OELSCHLAEGER T A.Mechanisms of probiotic actions-A review[J].Int J Med Microbiol,2010,300(1):57-62.
[26] 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.SHAO Q,HUANG Y J,HAN Y,et al.Structural composition,biological function of yeast cell wall and its application in aquaculture[J]. Zhejiang Journal Animal Science and Veterinary Medicine,2017,42(1):13-16.(in Chinese)
[27] XIAO Z G,TRINCADO C A,MURTAUGH M P.β-Glucan enhancement of T cell IFNγ response in swine[J].Vet Immunol Immunopathol, 2004,102(3):315-320.
[28] 刘红芝,王强,周素梅.酵母甘露聚糖分离提取及功能活性研究进展[J].食品科学,2008,29(5):465-468.LIU H Z,WANG Q,ZHOU S M.Research progress on extraction and activated function of yeast Mannan[J].Food Science, 2008, 29(5): 465-468.(in Chinese)
[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(5):4-8.XU H,SHEN L Q,KANG K,et al.Effect of supplementation of yeast cell wall in diets on performance of early lactating dairy cows[J]. China Dairy Cattle,2017(5):4-8.(in Chinese)
[2] PéREZ-RUCHEL A,REPETTO J L,CAJARUILLE C.Suitability of live yeast addition to alleviate the adverse effects due to the restriction of the time of access to feed in sheep fed only pasture[J].J Anim Physiol Anim Nutr (Berl),2013,97(6):1043-1050.
[3] GUEDES C M,GON?ALVES D,RODRIGUES M A M,et al.Effects of a Saccharomyces cerevisiae yeast on ruminal fermentation and fibre degradation of maize silages in cows[J].Anim Feed Sci Technol,2008,145(1-4):27-40.
[4] 陈惠娴,毛若雨,滕达,等.二硫键及其连接方式对防御素抗菌功能的影响研究进展[J].生物技术通报,2016,32(9):32-37.CHEN H X,MAO R Y,TENG D,et al.Review on effects of disulfide bond and its connection on antimicrobial activity in defensins[J]. Biotechnology Bulletin,2016,32(9):32-37.(in Chinese)
[5] ZHAO P W,CAO G F.Production of bioactive sheep β-defensin-1 in Pichia pastoris[J].J Ind Microbiol Biotechnol, 2012, 39(1): 11-17.
[6] XIAO Y J,HUGHES A L,ANDO J,et al.A genome-wide screen identifies a single β-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins[J].BMC Genomics,2004,5(1):56.
[7] MONTELEONE G,CALASCIBETTA D,SCATURROM,et al.Polymorphisms of β-defensin genes in Valle del Belice dairy sheep[J]. Mol Biol Rep,2011,38(8):5405-5412.
[8] 李砚,杨银凤.家畜体内防御素多态性和表达的研究进展[J].中国畜牧兽医,2013,40(3):160-168.LI Y,YANG Y F.Research progress of polymorphism and expression of defensins in vivo of livestock[J].China Animal Husbandry & Veterinary Medicine,2013,40(3):160-168.(in Chinese)
[9] 黎观红,洪智敏,贾永杰,等.抗菌肽的抗菌作用及其机制[J].动物营养学报,2011,23(4):546-555.LI G H,HONG Z M,JIA Y J,et al.Activities and mechanisms of action of antimicrobial peptides[J].Chinese Journal of Animal Nutrition, 2011,23(4):546-555.(in Chinese)
[10] SEMPLE F,DORIN J R.β-defensins:multifunctional modulators of infection,inflammation and more?[J].J Innate Immun, 2012, 4(4): 337-348.
[11] DASGUPTA T,NWEZE E I,YUE H,et al.Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53[J].Oncotarget,2016,7(19):27430-27444.
[12] CORLEIS B,LISANTI A C,K?RNER C,et al.Early type I Interferon response induces upregulation of human β-defensin 1 during acute HIV-1 infection[J].PLoS One,2017,12(3):e0173161.
[13] RYAN L K,DAI J H,YIN Z W,et al.Modulation of human β-defensin-1 (hBD-1) in plasmacytoid dendritic cells (PDC),monocytes,and epithelial cells by influenza virus,Herpes simplex virus,and Sendai virus and its possible role in innate immunity[J]. J Leukoc Biol,2011,90(2):343-356.
[14] 李砚.植物乳杆菌对绵羊瘤胃上皮细胞内SBD-1基因表达的影响[D].内蒙古:内蒙古农业大学,2013.LI Y.Effect of Lactobacillus plantarum on the expression of SBD-1 gene in rumen epithelial cells of sheep[D].Inner Mongolia:Inner Mongolia Agricultural University, 2013.(in Chinese)
[15] 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)
[16] JIN X,ZHANG M,ZHU X M,et al.Modulation of ovine SBD-1 expression by Saccharomyces cerevisiae in ovine ruminal epithelial cells[J].BMC Vet Res,2018,14(1):134.
[17] COSTA M O,HARDING J C,HILL J E.Development and evaluation of a porcine in vitro colon organ culture technique[J].in vitro Cell Dev Biol Anim,2016,52(9):942-952.
[18] SCHMITTGEN T D,ZAKRAJSEK B A.Effect of experimental treatment on housekeeping gene expression:validation by real-time,quantitative RT-PCR[J].J Biochem Biophysical Methods,2000,46(1-2):69-81.
[19] NAVA G M,ESCORCIA M,CASTA?EDA M P.Molecular diversity of the antimicrobial domain of Beta-defensin 3 and homologous peptides[J].Comp Funct Genomics,2009,2009:Article ID 983636.
[20] WANG S,ZENG X F,YANG Q,et al.Antimicrobial peptides as potential alternatives to antibiotics in food animal industry[J].Int J Mol Sci,2016,17(5):603.
[21] 田巧珍,金鑫,张曼,等.酿酒酵母细胞壁诱导体外培养绵羊瘤胃上皮细胞表达SBD-1的研究[J].畜牧兽医学报,2018,49(5):927-934.TIAN Q Z,JIN X,ZHANG M,et al.Effects of cell wall of Saccharomyces cerevisiae on the expression of SBD-1 in cultured ruminal epithelial cells of sheep[J].Acta Veterinaria et Zootechnica Sinica,2018,49(5):927-934.(in Chinese)
[22] SCHLEE M,HARDER J,K?TEN B,et al.Probiotic lactobacilli and VSL#3 induce enterocyte β-defensin 2[J].Clin Exp Immunol, 2008, 151(3):528-535.
[23] 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.LI G H,HONG Z M,JIA Y J,et al.Effect of Lactobacillus rhamnosus LGA on β-defensin 9 expression in cultured chicken small intestinal epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2012,43(4):634-641.(in Chinese)
[24] 范燕茹,王佩,金鑫,等.乳酸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.FAN Y R,WANG P,JIN X,et al.The expression of SBD-1 in the rumen epithelium cells of sheep induced by lactobacillus[J].Chinese Journal of Veterinary Science,2016,36(4):604-612.(in Chinese)
[25] OELSCHLAEGER T A.Mechanisms of probiotic actions-A review[J].Int J Med Microbiol,2010,300(1):57-62.
[26] 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.SHAO Q,HUANG Y J,HAN Y,et al.Structural composition,biological function of yeast cell wall and its application in aquaculture[J]. Zhejiang Journal Animal Science and Veterinary Medicine,2017,42(1):13-16.(in Chinese)
[27] XIAO Z G,TRINCADO C A,MURTAUGH M P.β-Glucan enhancement of T cell IFNγ response in swine[J].Vet Immunol Immunopathol, 2004,102(3):315-320.
[28] 刘红芝,王强,周素梅.酵母甘露聚糖分离提取及功能活性研究进展[J].食品科学,2008,29(5):465-468.LIU H Z,WANG Q,ZHOU S M.Research progress on extraction and activated function of yeast Mannan[J].Food Science, 2008, 29(5): 465-468.(in Chinese)
[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(5):4-8.XU H,SHEN L Q,KANG K,et al.Effect of supplementation of yeast cell wall in diets on performance of early lactating dairy cows[J]. China Dairy Cattle,2017(5):4-8.(in Chinese)
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