酿酒酵母甘露聚糖诱导绵羊瘤胃上皮细胞SBD-1表达的信号通路研究
|
摘要
β-防御素是机体的内源性抗菌肽。酿酒酵母(Saccharomyces cerevisiae)甘露聚糖能够诱导绵羊(Ovis aries)瘤胃上皮细胞(ruminal epithelial cells, RECs)β-防御素-1 (sheepβ-defensin-1, SBD-1)的表达,但是诱导机制尚不明确,限制了甘露聚糖制剂的开发利用。为了探讨丝裂原活化蛋白激酶(mitogenactivated protein kinase, MAPK)信号传导通路(p38, ERK1/2, JNK)和核因子κB (nuclear factorκB, NF-κB)通路是否参与甘露聚糖诱导绵羊RECs SBD-1的表达,首先利用qPCR和Western blot方法检测甘露聚糖对p38、ERK1/2、JNK和NF-κB表达的影响;然后用甘露聚糖分别刺激RECs 5、15、30、45和60 min后,通过Western blot检测p38、ERK1/2、JNK、IκB和p65的磷酸化水平;最后采用qPCR和ELISA方法检测p38、ERK1/2、JNK和NF-κB的特异性抑制剂对甘露聚糖诱导SBD-1表达的影响。结果显示,甘露聚糖刺激使RECs中p38、ERK1/2、JNK和NF-κB的m RNA水平以及p38、JNK、ERK1/2、IκB和p65的磷酸化水平均显著提高(P<0.01);同时,甘露聚糖刺激RECs不同时间可以使p38、ERK1/2、JNK、IκB和p65发生磷酸化;此外,p38、ERK1/2、JNK和NF-κB的抑制剂均能极显著降低甘露聚糖诱导SBD-1的表达(P<0.01),且p38抑制剂的抑制效果最明显。上述结果提示,酿酒酵母甘露聚糖诱导绵羊RECs SBD-1表达的过程可能由p38、ERK1/2、JNK和NF-κB信号通路共同调节,并且p38信号通路可能发挥更主要的作用。该研究探讨了甘露聚糖诱导防御素表达的机理,为解释甘露聚糖促免疫功能提供了新线索,也为更好地开发和利用甘露聚糖制剂提供了参考依据。
β-defensins are the endogenous antibacterial peptide of the organism. Saccharomyces cerevisiae mannan can induce ovine Ruminal epithelial cells(RECs) β-defensin-1(SBD-1) expression. However, its mechanism of inducing SBD-1 expression is unclear, which limits the development and utilization of mannan preparations. In order to explore whether mitogen-activated protein kinase(MAPK)(p38, ERK1/2, JNK) and Nuclear factor κB(NF-κB) are involved in the expression of SBD-1 in ovine RECs induced by mannan.Firstly, the effects of mannan on the expression of p38, ERK1/2, JNK and NF-κB were examined by qPCR and Western blot. Then the phosphorylation levels of p38, ERK1/2, JNK, IκB and p65 were detected by Western blot after stimulation of RECs with mannan for 5, 15, 30, 45 and 60 min, respectively. Finally, qPCR and ELISA were used to detect the effects of specific inhibitors of p38, ERK1/2, JNK and NF-κB on mannaninduced SBD-1 expression to determine the involvement of p38, ERK1/2, JNK and NF-κB pathways in the expression of SBD-1 induced by mannan. The results showed that mannan stimulation significantly increased the mRNA levels of p38, ERK1/2, JNK and NF-κB in RECs and the phosphorylation levels of p38, JNK,ERK1/2, IκB and p65(P<0.01). And mannan stimulated RECs different times and activated phosphorylation of p38, ERK1/2, JNK, IκB and p65. Moreover, treatment with p38, ERK1/2, JNK and NF-κB inhibitors significantly decreased the expression of SBD-1 induced by mannan(P<0.01), and the inhibitory effect of p38 inhibitor on SBD-1 was the most obvious. These results indicate that the S. cerevisiae mannan-induced SBD-1 expression in RECs is mediated by MAPK and NF-κB signaling pathways, and the p38 signaling pathway may play a major role. This study explains the function of mannan to promote immunity from the mechanism of mannan-induced defensin expression, and provides a theoretical basis for better development and utilization of mannan preparations.
β-defensins are the endogenous antibacterial peptide of the organism. Saccharomyces cerevisiae mannan can induce ovine Ruminal epithelial cells(RECs) β-defensin-1(SBD-1) expression. However, its mechanism of inducing SBD-1 expression is unclear, which limits the development and utilization of mannan preparations. In order to explore whether mitogen-activated protein kinase(MAPK)(p38, ERK1/2, JNK) and Nuclear factor κB(NF-κB) are involved in the expression of SBD-1 in ovine RECs induced by mannan.Firstly, the effects of mannan on the expression of p38, ERK1/2, JNK and NF-κB were examined by qPCR and Western blot. Then the phosphorylation levels of p38, ERK1/2, JNK, IκB and p65 were detected by Western blot after stimulation of RECs with mannan for 5, 15, 30, 45 and 60 min, respectively. Finally, qPCR and ELISA were used to detect the effects of specific inhibitors of p38, ERK1/2, JNK and NF-κB on mannaninduced SBD-1 expression to determine the involvement of p38, ERK1/2, JNK and NF-κB pathways in the expression of SBD-1 induced by mannan. The results showed that mannan stimulation significantly increased the mRNA levels of p38, ERK1/2, JNK and NF-κB in RECs and the phosphorylation levels of p38, JNK,ERK1/2, IκB and p65(P<0.01). And mannan stimulated RECs different times and activated phosphorylation of p38, ERK1/2, JNK, IκB and p65. Moreover, treatment with p38, ERK1/2, JNK and NF-κB inhibitors significantly decreased the expression of SBD-1 induced by mannan(P<0.01), and the inhibitory effect of p38 inhibitor on SBD-1 was the most obvious. These results indicate that the S. cerevisiae mannan-induced SBD-1 expression in RECs is mediated by MAPK and NF-κB signaling pathways, and the p38 signaling pathway may play a major role. This study explains the function of mannan to promote immunity from the mechanism of mannan-induced defensin expression, and provides a theoretical basis for better development and utilization of mannan preparations.
引文
段德鉴,蒋献,冉玉平,等.2005.白念珠菌对角质形成细胞β防御素-2 m RNA表达的影响[J].中华皮肤科杂志,38(4):202-204.(Duan D J,Jiang X,Ran Y P,et al.2005.The effect of Candida albicans on the expression level of humanβ-defensin-2 mRNA in keratinocytes in vitro[J].Chinese Journal of Dermatology,38(4):202-204.)
李砚,杨银凤.2013.家畜体内防御素的多态性和表达[J].中国畜牧兽医,40(3):160-168.(Li Y,Yang Y F.2013.Research progress of polymorphism and expression of defensins in vivo of livestock[J].China Animal Husbandry&Veterinary Medicine,40(3):160-168.)
盛金良,陈创夫,杨霞.等.2009.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,40(1):20-25.(Sheng J L,Chen C F,Yang X.et al.2009.Distribution and quantitative assay of sheepβ-defensin-1 mRNA in differently developmental stages[J].Acta Veterinaria et Zootechnica Sinica,40(1):20-25.)
Ackermann M R,Gallup J M,Zabner J,et al.2004.Differential expression of sheepβ-defensin-1 and-2 and interleukin 8 during acute Mannheimia haemolytica pneumonia[J].Microbial Pathogenesis,37(1):21-27.
Barabas N,R?hrl J,Holler E,et al.2013.Beta-defensins activate macrophages and synergize in pro-inflammatory cytokine expression induced by TLR ligands[J].Immunobiology,218(7):1005-1011.
Brogden K A,Ackermann M,McCray P B,et al.2013.Antimicrobial peptides in animals and their role in host defences[J].International Journal of Antimicrobial Agents,22(5):465-478.
Campoverde C,Milne D J,Estevez A,et al.2017.Ontogeny and modulation,after PAMPs stimulation,ofβ-defensin,hepcidin,and piscidin antimicrobial peptides in meagre(Argyrosomus regius)[J].Fish&Shellfish Immunology,69:200-210.
Cohen-Kedar S,Baram L,Elad H,et al.2015.Human intestinal epithelial cells respond toβ-glucans via Dectin-1and Syk[J].European Journal of Immunology,44(12):3729-3740.
Cuperus T,Coorens M,van Dijk A,et al.2013.Avian host defense peptides[J].Developmental and Comparative Immunology,41(3):352-369.
Drummond R A,Saijo S,Iwakura Y,et al.2011.The role of Syk/CARD9 coupled C-type lectins in antifungal immunity[J].European Journal of Immunology,41(2):276-281.
Gácser A,Tiszlavicz Z,Németh T,et al.2014.Induction of human defensins by intestinal Caco-2 cells after interactions with opportunistic Candida species[J].Microbes and Infection,16(1):80-85.
Gan Y,Cui X,Ma T,et al.2014.Paeoniflorin upregulatesβ-defensin-2 expression in human bronchial epithelial cell through the p38 MAPK,ERK,and NF-κB signaling pathways[J].Inflammation,37(5):1468-1475.
Garcia D T,Ferriani B A,Jacovaci F A,et al.2018.Inclusion of live yeast and mannan-oligosaccharides in high grainbased diets for sheep:Ruminal parameters,inflammatory response and rumen morphology[J].PLoS One,13(2):e0193313.
Huttner K M,Brezinski-Caliguri D J,Mahoney M M,et al.1998.Antimicrobial peptide expression is developmentally regulated in the ovine gastrointestinal tract[J].Journal of Nutrition,128(2):297-299.
Huttner K M,Lambeth M R,Burkin H R,et al.1998.Localization and genomic organization of sheep antimicrobial peptide genes[J].Gene,206(1):85-91.
Jiménez-Garcia L,Herránz S,Luque A,et al.2015.Critical role of p38 MAPK in IL-4-induced alternative activation of peritoneal macrophages[J].European Journal of Immunology,45(1):273-286.
Jin X,Zhang M,Zhu X M,et al.2018.Modulation of ovine SBD-1 expression by Saccharomyces cerevisiae in ovine ruminal epithelial cells[J].BMC Veterinary Research,14(1):134.
Kim J H,Kim K H,Kim H J,et al.2015.Expression of betadefensin 131 promotes an innate immune response in human prostate epithelial cells[J].PLoS One,10(12):e0144776.
Li Q,Bao F,Zhi D,et al.2016.Lipopolysaccharide induces SBD-1 expression via the P38 MAPK signaling pathway in ovine oviduct epithelial cells[J].Lipids in Health and Disease,15(1):127.
Medeiros M C,Frasnelli S C,Bastos Ade S,et al.2014.Modulation of cell proliferation,survival and gene expression by RAGE and TLR signaling in cells of the innate and adaptive immune response:Role of p38 MAPK and NF-κB[J].Journal of Applied Oral Science,22(3):185-193.
Mitchell D,Yong M,Raju J,et al.2011.Toll-like receptor-mediated adjuvanticity and immunomodulation in dendritic cells:Implications for peptide vaccines[J].Human vaccines&immunotherapeutics,7 Suppl:85-93.
Oelschlaeger T A.2010.Mechanisms of probiotic actions-Areview[J].International Journal of Medical Microbiology,300(1):57-62.
Ouellette A J.2006.Paneth cell alpha-defensin synthesis and function[J].Current Topics in Microbiology and Immunology,306:1-25.
Oyanedel D,Gonzalez R,Brokordt K,et al.2016.Insight into the messenger role of reactive oxygen intermediates in immunostimulated hemocytes from the scallop Argopec?ten purpuratus[J].Developmental&Comparative Immunology,65:226-230.
Pontón J,Omaetxebarría M J,Elguezabal N,et al.2001.Immunoreactivity of the fungal cell wall[J].Medical Mycology,39 Suppl 1:101-110.
Robinson M J,Osorio F,Rosas M,et al.2009.Dectin-2 is a Syk-coupled pattern recognition receptor crucial for Th17 responses to fungal infection[J].Journal of Experimental Medicine,206(9):2037-2051.
Saijo S,Ikeda S,Yamabe K,et al.2010.Dectin-2 recognition of alpha-mannans and induction of Th17 cell differentiation is essential for host defense against Candida albi?cans[J].Immunity,32(5):681-691.
Shao Y,Wang Z,Tian X,et al.2016.Yeastβ-d-glucans induced antimicrobial peptide expressions against Salmo?nella infection in broiler chickens[J].International Journal of Biological Macromolecules,85:573-584.
Shao Z J,Zheng X W,Feng T,et al.2012.Andrographolide exerted its antimicrobial effects by upregulation of humanβ-defensin-2 induced through p38 MAPK and NF-κB pathway in human lung epithelial cells[J].Canadian Journal of Physiology and Pharmacology,90(5):647-653.
Shilo B Z.2014.The regulation and functions of MAPK pathways in Drosophila[J].Methods,68(1):151-159.
Wang W,Li Z,Han Q,et al.2016.Dietary live yeast and mannan-oligosaccharide supplementation attenuate intestinal inflammation and barrier dysfunction induced by Escherichia coli in broilers[J].British Journal of Nutrition,116(11):1878-1888.
Wen S,Cao G,Bao T,et al.2012.Modulation of ovine SBD-1 expression by 17 beta-estradiol in ovine oviduct epithelial cells[J].BMC Veterinary Research,8:143.
Yang S H,Sharrocks A D,Whitmarsh A J.2013.MAP kinase signalling cascades and transcriptional regulation[J].Gene,513(1):1-13
Zhang M,Jin X,Yang Y F.2018.β-glucans from Saccharomy?ces cerevisiae induce SBD-1 production in ovine ruminal epithelial cells via the Dectin-1-Syk-NF-κB signaling pathway[J].Cellular Signalling,53:304-315.
李砚,杨银凤.2013.家畜体内防御素的多态性和表达[J].中国畜牧兽医,40(3):160-168.(Li Y,Yang Y F.2013.Research progress of polymorphism and expression of defensins in vivo of livestock[J].China Animal Husbandry&Veterinary Medicine,40(3):160-168.)
盛金良,陈创夫,杨霞.等.2009.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,40(1):20-25.(Sheng J L,Chen C F,Yang X.et al.2009.Distribution and quantitative assay of sheepβ-defensin-1 mRNA in differently developmental stages[J].Acta Veterinaria et Zootechnica Sinica,40(1):20-25.)
Ackermann M R,Gallup J M,Zabner J,et al.2004.Differential expression of sheepβ-defensin-1 and-2 and interleukin 8 during acute Mannheimia haemolytica pneumonia[J].Microbial Pathogenesis,37(1):21-27.
Barabas N,R?hrl J,Holler E,et al.2013.Beta-defensins activate macrophages and synergize in pro-inflammatory cytokine expression induced by TLR ligands[J].Immunobiology,218(7):1005-1011.
Brogden K A,Ackermann M,McCray P B,et al.2013.Antimicrobial peptides in animals and their role in host defences[J].International Journal of Antimicrobial Agents,22(5):465-478.
Campoverde C,Milne D J,Estevez A,et al.2017.Ontogeny and modulation,after PAMPs stimulation,ofβ-defensin,hepcidin,and piscidin antimicrobial peptides in meagre(Argyrosomus regius)[J].Fish&Shellfish Immunology,69:200-210.
Cohen-Kedar S,Baram L,Elad H,et al.2015.Human intestinal epithelial cells respond toβ-glucans via Dectin-1and Syk[J].European Journal of Immunology,44(12):3729-3740.
Cuperus T,Coorens M,van Dijk A,et al.2013.Avian host defense peptides[J].Developmental and Comparative Immunology,41(3):352-369.
Drummond R A,Saijo S,Iwakura Y,et al.2011.The role of Syk/CARD9 coupled C-type lectins in antifungal immunity[J].European Journal of Immunology,41(2):276-281.
Gácser A,Tiszlavicz Z,Németh T,et al.2014.Induction of human defensins by intestinal Caco-2 cells after interactions with opportunistic Candida species[J].Microbes and Infection,16(1):80-85.
Gan Y,Cui X,Ma T,et al.2014.Paeoniflorin upregulatesβ-defensin-2 expression in human bronchial epithelial cell through the p38 MAPK,ERK,and NF-κB signaling pathways[J].Inflammation,37(5):1468-1475.
Garcia D T,Ferriani B A,Jacovaci F A,et al.2018.Inclusion of live yeast and mannan-oligosaccharides in high grainbased diets for sheep:Ruminal parameters,inflammatory response and rumen morphology[J].PLoS One,13(2):e0193313.
Huttner K M,Brezinski-Caliguri D J,Mahoney M M,et al.1998.Antimicrobial peptide expression is developmentally regulated in the ovine gastrointestinal tract[J].Journal of Nutrition,128(2):297-299.
Huttner K M,Lambeth M R,Burkin H R,et al.1998.Localization and genomic organization of sheep antimicrobial peptide genes[J].Gene,206(1):85-91.
Jiménez-Garcia L,Herránz S,Luque A,et al.2015.Critical role of p38 MAPK in IL-4-induced alternative activation of peritoneal macrophages[J].European Journal of Immunology,45(1):273-286.
Jin X,Zhang M,Zhu X M,et al.2018.Modulation of ovine SBD-1 expression by Saccharomyces cerevisiae in ovine ruminal epithelial cells[J].BMC Veterinary Research,14(1):134.
Kim J H,Kim K H,Kim H J,et al.2015.Expression of betadefensin 131 promotes an innate immune response in human prostate epithelial cells[J].PLoS One,10(12):e0144776.
Li Q,Bao F,Zhi D,et al.2016.Lipopolysaccharide induces SBD-1 expression via the P38 MAPK signaling pathway in ovine oviduct epithelial cells[J].Lipids in Health and Disease,15(1):127.
Medeiros M C,Frasnelli S C,Bastos Ade S,et al.2014.Modulation of cell proliferation,survival and gene expression by RAGE and TLR signaling in cells of the innate and adaptive immune response:Role of p38 MAPK and NF-κB[J].Journal of Applied Oral Science,22(3):185-193.
Mitchell D,Yong M,Raju J,et al.2011.Toll-like receptor-mediated adjuvanticity and immunomodulation in dendritic cells:Implications for peptide vaccines[J].Human vaccines&immunotherapeutics,7 Suppl:85-93.
Oelschlaeger T A.2010.Mechanisms of probiotic actions-Areview[J].International Journal of Medical Microbiology,300(1):57-62.
Ouellette A J.2006.Paneth cell alpha-defensin synthesis and function[J].Current Topics in Microbiology and Immunology,306:1-25.
Oyanedel D,Gonzalez R,Brokordt K,et al.2016.Insight into the messenger role of reactive oxygen intermediates in immunostimulated hemocytes from the scallop Argopec?ten purpuratus[J].Developmental&Comparative Immunology,65:226-230.
Pontón J,Omaetxebarría M J,Elguezabal N,et al.2001.Immunoreactivity of the fungal cell wall[J].Medical Mycology,39 Suppl 1:101-110.
Robinson M J,Osorio F,Rosas M,et al.2009.Dectin-2 is a Syk-coupled pattern recognition receptor crucial for Th17 responses to fungal infection[J].Journal of Experimental Medicine,206(9):2037-2051.
Saijo S,Ikeda S,Yamabe K,et al.2010.Dectin-2 recognition of alpha-mannans and induction of Th17 cell differentiation is essential for host defense against Candida albi?cans[J].Immunity,32(5):681-691.
Shao Y,Wang Z,Tian X,et al.2016.Yeastβ-d-glucans induced antimicrobial peptide expressions against Salmo?nella infection in broiler chickens[J].International Journal of Biological Macromolecules,85:573-584.
Shao Z J,Zheng X W,Feng T,et al.2012.Andrographolide exerted its antimicrobial effects by upregulation of humanβ-defensin-2 induced through p38 MAPK and NF-κB pathway in human lung epithelial cells[J].Canadian Journal of Physiology and Pharmacology,90(5):647-653.
Shilo B Z.2014.The regulation and functions of MAPK pathways in Drosophila[J].Methods,68(1):151-159.
Wang W,Li Z,Han Q,et al.2016.Dietary live yeast and mannan-oligosaccharide supplementation attenuate intestinal inflammation and barrier dysfunction induced by Escherichia coli in broilers[J].British Journal of Nutrition,116(11):1878-1888.
Wen S,Cao G,Bao T,et al.2012.Modulation of ovine SBD-1 expression by 17 beta-estradiol in ovine oviduct epithelial cells[J].BMC Veterinary Research,8:143.
Yang S H,Sharrocks A D,Whitmarsh A J.2013.MAP kinase signalling cascades and transcriptional regulation[J].Gene,513(1):1-13
Zhang M,Jin X,Yang Y F.2018.β-glucans from Saccharomy?ces cerevisiae induce SBD-1 production in ovine ruminal epithelial cells via the Dectin-1-Syk-NF-κB signaling pathway[J].Cellular Signalling,53:304-315.