脾酪氨酸激酶参与酿酒酵母甘露聚糖诱导绵羊瘤胃上皮细胞β-防御素-1(SBD-1)的表达
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摘要
为了探索脾酪氨酸激酶(Syk)是否参与酿酒酵母甘露聚糖(S.c M)诱导绵羊瘤胃上皮细胞(ORECs)β-防御素-1(SBD-1)表达的过程,首先利用免疫组化、RT-PCR和免疫荧光等方法检测Syk在ORECs内的表达情况;然后采用qPCR和Western blot方法检测S.c M刺激ORECs后Syk的表达变化,同时用Western blot方法检测Syk的磷酸化水平;接着用3条Syk特异性siRNAs(#1、#2和#3)转染ORECs 24 h后,qPCR检测Syk mRNA的表达变化,筛选出干扰效果最佳的Syk siRNA;最后用效果最佳的siRNA和Syk特异性抑制剂R406分别处理ORECs后,采用qPCR和ELISA检测SBD-1的表达变化,以确定Syk在S.c M诱导SBD-1表达过程中的作用。结果显示:Syk在ORECs内表达;且S.c M刺激ORECs后Syk的mRNA和蛋白表达水平显著高于未刺激组(P<0.01或P<0.05),S.c M刺激ORECs不同时间(5、15、30、45和60 min)均能使Syk发生磷酸化,且刺激15 min后磷酸化水平达到最大(P<0.01);此外,Syk的3条特异性siRNAs转染ORECs后Syk的表达均降低,且Syk siRNA#2的抑制效果最明显(P<0.01);同时Syk siRNA#2和R406均能极显著降低S.c M诱导ORECs SBD-1的表达(P<0.01)。上述结果表明,Syk参与S.c M诱导ORECs SBD-1的表达。
The aim of this study was to explore whether spleen tyrosine kinase(Syk) is involved in the expression of β-defensin-1(SBD-1) in ovine ruminal epithelial cells(ORECs) induced by Saccharomyces cerevisiae Mannan(S.c M). Firstly, the expression of Syk in rumen tissues and ORECs was detected by immunohistochemistry, RT-PCR and immunofluorescence. Then, qPCR and Western blot were used to detect the expression of Syk after S.c M stimulation of ORECs, and the phosphorylation level of Syk was detected by Western blot. The ORECs were transfected with three Syk-specific siRNAs(#1, #2 and #3) for 24 h, qPCR was used to detect the expression of Syk mRNA, and the Syk siRNA with the best interference effect was screened. Finally, after treatment of ORECs with the best siRNA and Syk specific inhibitor R406, the expression of SBD-1 was detected by qPCR and ELISA to determine the role of Syk in the expression of SBD-1 induced by S.c M. The results showed that Syk was expressed in ORECs, and the expression level of Syk mRNA and protein were significantly higher in mannan-stimulated ORECs than in unstimulated group(P<0.05). After S.c M stimulation of ORECs for different time(5, 15, 30, 45 and 60 min), Syk was phosphorylated, and the phosphorylation level reached the maximum after 15 min stimulation(P<0.01). In addition, the expression of Syk was decreased after Syk's three specific siRNAs transfected with ORECs, and the inhibitory effect of Syk siRNA#2 was the most significant(P<0.01). Simultaneously, Syk siRNA#2 and R406 significantly reduced S.c M-induced ORECs. Expression of SBD-1(P<0.01). The above results indicate that Syk is involved in the expression of SBD-1 induced by S.c M.
The aim of this study was to explore whether spleen tyrosine kinase(Syk) is involved in the expression of β-defensin-1(SBD-1) in ovine ruminal epithelial cells(ORECs) induced by Saccharomyces cerevisiae Mannan(S.c M). Firstly, the expression of Syk in rumen tissues and ORECs was detected by immunohistochemistry, RT-PCR and immunofluorescence. Then, qPCR and Western blot were used to detect the expression of Syk after S.c M stimulation of ORECs, and the phosphorylation level of Syk was detected by Western blot. The ORECs were transfected with three Syk-specific siRNAs(#1, #2 and #3) for 24 h, qPCR was used to detect the expression of Syk mRNA, and the Syk siRNA with the best interference effect was screened. Finally, after treatment of ORECs with the best siRNA and Syk specific inhibitor R406, the expression of SBD-1 was detected by qPCR and ELISA to determine the role of Syk in the expression of SBD-1 induced by S.c M. The results showed that Syk was expressed in ORECs, and the expression level of Syk mRNA and protein were significantly higher in mannan-stimulated ORECs than in unstimulated group(P<0.05). After S.c M stimulation of ORECs for different time(5, 15, 30, 45 and 60 min), Syk was phosphorylated, and the phosphorylation level reached the maximum after 15 min stimulation(P<0.01). In addition, the expression of Syk was decreased after Syk's three specific siRNAs transfected with ORECs, and the inhibitory effect of Syk siRNA#2 was the most significant(P<0.01). Simultaneously, Syk siRNA#2 and R406 significantly reduced S.c M-induced ORECs. Expression of SBD-1(P<0.01). The above results indicate that Syk is involved in the expression of SBD-1 induced by S.c M.
引文
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[2] BARABAS N,R?HRL J,HOLLER E,et al.Beta-defensins activate macrophages and synergize in pro-inflammatory cytokine expression induced by TLR ligands[J].Immunobiology,2013,218(7):1005-1011.
[3] CUPERUS T,COORENS M,VAN DIJK A,et al.Avian host defense peptides[J].Dev Comp Immunol,2013,41(3):352-369.
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[6] 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.
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[8] 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)
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[10] WANG W W,LI Z,HAN Q Q,et al.Dietary live yeast and mannan-oligosaccharide supplementation attenuate intestinal inflammation and barrier dysfunction induced by Escherichia coli in broilers[J].Br J Nutr,2016,116(11):1878-1888.
[11] 段德鉴,蒋献,冉玉平,等.白念珠菌对角质形成细胞β防御素-2mRNA表达的影响[J].中华皮肤科杂志,2005,38(4):202-204.DUAN D J,JIANG X,RAN Y P,et al.The effect of Candida albicans on the expression level of human β-defensin-2 mRNA in keratinocytes in vitro[J].Chinese Journal of Dermatology,2005,38(4):202-204.(in Chinese)
[12] 金鑫,张曼,王云鹤,等.酿酒酵母甘露聚糖对绵羊瘤胃上皮细胞β-防御素-1(SBD-1)表达的影响[J].中国预防兽医学报,2018,40(12):1117-1121.JIN X,ZHANG M,WANG Y H,et al.Effects of mannan on the expression of SBD-1 in sheep rumen epithelial cells[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(12):1117-1121.(in Chinese)
[13] KUROSAKI T,TAKATA M,YAMANASHI Y,et al.Syk activation by the Src-family tyrosine kinase in the B cell receptor signaling[J].J Exp Med,1994,179(5):1725-1729.
[14] TSUCHIDA S,YANAGI S,INATOME R,et al.Purification of a 72-kDa protein-tyrosine kinase from rat liver and its identification as Syk:involvement of Syk in signaling events of hepatocytes[J].J Biochem,2000,127(2):321-327.
[15] OKAMURA S,NG C C,KOYAMA K,et al.Identification of seven genes regulated by wild-type p53 in a colon cancer cell line carrying a well-controlled wild-type p53 expression system[J].Oncol Res,1999,11(6):281-285.
[16] COOPMAN P J P,DO M T H,BARTH M,et al.The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells[J].Nature,2000,406(6797):742-747.
[17] COHEN-KEDAR S,BARAM L,ELAD H,et al.Human intestinal epithelial cells respond to β-glucans via Dectin-1 and Syk[J].Eur J Immunol,2014,44(12):3729-3740.
[18] DRUMMOND R A,SAIJO S,IWAKURA Y,et al.The role of Syk/CARD9 coupled C-type lectins in antifungal immunity[J].Eur J Immunol,2011,41(2):276-281.
[19] TAKADA Y,AGGARWAL B B.TNF activates Syk protein tyrosine kinase leading to TNF-induced MAPK activation,NF-κB activation,and apoptosis[J].J Immunol,2004,173(2):1066-1077.
[20] KERRIGAN A M,BROWN G D.Syk-coupled C-type lectin receptors that mediate cellular activation via single tyrosine based activation motifs[J].Immunol Rev,2010,234(1):335-352.
[21] SADA K,TAKANO T,YANAGI S,et al.Structure and function of Syk protein-tyrosine kinase[J].J Biochem,2001,130(2):177-186.
[22] PATTERSON H,NIBBS R,MCINNES I,et al.Protein kinase inhibitors in the treatment of inflammatory and autoimmune diseases[J].Clin Exp Immunol,2014,176(1):1-10.
[23] PARK G B,KIM D.Cigarette smoke-induced EGFR activation promotes epithelial mesenchymal migration of human retinal pigment epithelial cells through regulation of the FAK-mediated Syk/Src pathway[J].Mol Med Rep,2018,17(3):3563-3574.
[24] LIU Y,ZHAO G Q,LIN J,et al.The role of Syk signaling in antifungal innate immunity of human corneal epithelial cells[J].BMC Ophthalmol,2015,15:55.
[25] YANG W S,KIM J S,HAN N J,et al.Toll-like receptor 4/spleen tyrosine kinase complex in high glucose signal transduction of proximal tubular epithelial cells[J].Cell Physiol Biochem,2015,35(6):2309-2319.
[26] ULANOVA M,PUTTAGUNTA L,MARCET-PALACIOS M,et al.Syk tyrosine kinase participates in β1-integrin signaling and inflammatory responses in airway epithelial cells[J].Am J Physiol-Lung Cell Mol Physiol,2005,288(3):L497-L507.
[27] PITCHER L A,VAN OERS N S C.T-cell receptor signal transmission:who gives an ITAM?[J].Trends Immunol,2003,24(10):554-560.
[28] YANG W S,SEO J W,HAN N J,et al.High glucose-induced NF-κB activation occurs via tyrosine phosphorylation of IκBα in human glomerular endothelial cells:involvement of Syk tyrosine kinase[J].Am J Physiol Renal Physiol-Ren Physiol,2008,294(5):F1065-F1075.
[2] BARABAS N,R?HRL J,HOLLER E,et al.Beta-defensins activate macrophages and synergize in pro-inflammatory cytokine expression induced by TLR ligands[J].Immunobiology,2013,218(7):1005-1011.
[3] CUPERUS T,COORENS M,VAN DIJK A,et al.Avian host defense peptides[J].Dev Comp Immunol,2013,41(3):352-369.
[4] GáCSER A,TISZLAVICZ Z,NéMETH T,et al.Induction of human defensins by intestinal Caco-2 cells after interactions with opportunistic Candida species[J].Microbes Infect,2014,16(1):80-85.
[5] 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.
[6] 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.
[7] GARCIA DIAZ T,FERRIANI BRANCO A,JACOVACI F A,et al.Inclusion of live yeast and mannan-oligosaccharides in high grain-based diets for sheep:ruminal parameters,inflammatory response and rumen morphology[J].PLoS One,2018,13(2):e0193313.
[8] 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)
[9] MAGNELLI P,CIPOLLO J F,ABEIJON C.A refined method for the determination of Saccharomyces cerevisiae cell wall composition and β-1,6-glucan fine structure[J].Anal Biochem,2002,301(1):136-150.
[10] WANG W W,LI Z,HAN Q Q,et al.Dietary live yeast and mannan-oligosaccharide supplementation attenuate intestinal inflammation and barrier dysfunction induced by Escherichia coli in broilers[J].Br J Nutr,2016,116(11):1878-1888.
[11] 段德鉴,蒋献,冉玉平,等.白念珠菌对角质形成细胞β防御素-2mRNA表达的影响[J].中华皮肤科杂志,2005,38(4):202-204.DUAN D J,JIANG X,RAN Y P,et al.The effect of Candida albicans on the expression level of human β-defensin-2 mRNA in keratinocytes in vitro[J].Chinese Journal of Dermatology,2005,38(4):202-204.(in Chinese)
[12] 金鑫,张曼,王云鹤,等.酿酒酵母甘露聚糖对绵羊瘤胃上皮细胞β-防御素-1(SBD-1)表达的影响[J].中国预防兽医学报,2018,40(12):1117-1121.JIN X,ZHANG M,WANG Y H,et al.Effects of mannan on the expression of SBD-1 in sheep rumen epithelial cells[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(12):1117-1121.(in Chinese)
[13] KUROSAKI T,TAKATA M,YAMANASHI Y,et al.Syk activation by the Src-family tyrosine kinase in the B cell receptor signaling[J].J Exp Med,1994,179(5):1725-1729.
[14] TSUCHIDA S,YANAGI S,INATOME R,et al.Purification of a 72-kDa protein-tyrosine kinase from rat liver and its identification as Syk:involvement of Syk in signaling events of hepatocytes[J].J Biochem,2000,127(2):321-327.
[15] OKAMURA S,NG C C,KOYAMA K,et al.Identification of seven genes regulated by wild-type p53 in a colon cancer cell line carrying a well-controlled wild-type p53 expression system[J].Oncol Res,1999,11(6):281-285.
[16] COOPMAN P J P,DO M T H,BARTH M,et al.The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells[J].Nature,2000,406(6797):742-747.
[17] COHEN-KEDAR S,BARAM L,ELAD H,et al.Human intestinal epithelial cells respond to β-glucans via Dectin-1 and Syk[J].Eur J Immunol,2014,44(12):3729-3740.
[18] DRUMMOND R A,SAIJO S,IWAKURA Y,et al.The role of Syk/CARD9 coupled C-type lectins in antifungal immunity[J].Eur J Immunol,2011,41(2):276-281.
[19] TAKADA Y,AGGARWAL B B.TNF activates Syk protein tyrosine kinase leading to TNF-induced MAPK activation,NF-κB activation,and apoptosis[J].J Immunol,2004,173(2):1066-1077.
[20] KERRIGAN A M,BROWN G D.Syk-coupled C-type lectin receptors that mediate cellular activation via single tyrosine based activation motifs[J].Immunol Rev,2010,234(1):335-352.
[21] SADA K,TAKANO T,YANAGI S,et al.Structure and function of Syk protein-tyrosine kinase[J].J Biochem,2001,130(2):177-186.
[22] PATTERSON H,NIBBS R,MCINNES I,et al.Protein kinase inhibitors in the treatment of inflammatory and autoimmune diseases[J].Clin Exp Immunol,2014,176(1):1-10.
[23] PARK G B,KIM D.Cigarette smoke-induced EGFR activation promotes epithelial mesenchymal migration of human retinal pigment epithelial cells through regulation of the FAK-mediated Syk/Src pathway[J].Mol Med Rep,2018,17(3):3563-3574.
[24] LIU Y,ZHAO G Q,LIN J,et al.The role of Syk signaling in antifungal innate immunity of human corneal epithelial cells[J].BMC Ophthalmol,2015,15:55.
[25] YANG W S,KIM J S,HAN N J,et al.Toll-like receptor 4/spleen tyrosine kinase complex in high glucose signal transduction of proximal tubular epithelial cells[J].Cell Physiol Biochem,2015,35(6):2309-2319.
[26] ULANOVA M,PUTTAGUNTA L,MARCET-PALACIOS M,et al.Syk tyrosine kinase participates in β1-integrin signaling and inflammatory responses in airway epithelial cells[J].Am J Physiol-Lung Cell Mol Physiol,2005,288(3):L497-L507.
[27] PITCHER L A,VAN OERS N S C.T-cell receptor signal transmission:who gives an ITAM?[J].Trends Immunol,2003,24(10):554-560.
[28] YANG W S,SEO J W,HAN N J,et al.High glucose-induced NF-κB activation occurs via tyrosine phosphorylation of IκBα in human glomerular endothelial cells:involvement of Syk tyrosine kinase[J].Am J Physiol Renal Physiol-Ren Physiol,2008,294(5):F1065-F1075.