重组牛脂多糖结合蛋白对脂多糖诱导的奶牛乳腺炎症反应指标的影响
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摘要
[目的]本试验旨在研究不同剂量的重组牛脂多糖结合蛋白(rb LBP)作用于脂多糖(LPS)诱导的奶牛乳腺炎模型,探讨rb LBP对奶牛乳腺炎症反应的影响及其作用机制。[方法]选4头泌乳中后期、体质量为(500±25)kg的健康经产荷斯坦奶牛,采用4×4拉丁方设计,试验分为对照组、LPS(0.2μg·kg-1)模型组、低剂量rb LBP(1μg)处理组、高剂量rb LBP(20μg)处理组,分4期进行,每期15 d。[结果]灌注LPS后,奶牛发生乳腺炎症反应;与LPS模型组相比,低剂量rb LBP加剧LPS诱导的炎症反应,表现为组织髓过氧化物酶(MPO)活性明显升高(P<0.05),炎性因子TNF-α、IL-1β、IL-6的基因及蛋白表达量显著升高(P<0.05),TLR4和NF-κB的表达明显上调(P<0.05);而高剂量rb LBP有效缓解LPS诱导的炎症反应,表现为组织MPO活性明显降低(P<0.05),炎性因子TNF-α、IL-1β、IL-6表达量显著降低(P<0.05),TLR4和NF-κB的表达显著下调(P<0.05)。[结论]低剂量rb LBP能够增加LPS诱导的奶牛乳腺炎症反应,而高剂量rb LBP可以有效缓解LPS诱导的奶牛乳腺炎,其作用机制可能是通过激活TLR4/NF-κB信号通路从而影响炎性因子的转录表达。
[Objectives]This experiment was conducted to study the effect of recombinant bovine lipopolysaccharide(LPS)-binding protein(rb LBP) on LPS-induced bovine mastitis and to explore its relevant mechanism whether associated with the activation of TLR4/NF-κB signaling pathway. [Methods]Four lactating Holstein cows(500±25) kg were used in a 4× 4 latin square design and they were divided into the control group,LPS model group infused with LPS and the rb LBP treatment group infused with LPS and different doses of rb LBP concurrently. The experiment was conducted in four phases(15 days per phase). [Results]The results showed that inflammation was induced in mammary gland after infusing with LPS,and compared with the LPS model group,low dose of LBP(1 μg) enhanced the inflammatory response in mammary gland as indicated by the increased MPO activity and by the increased expression of TNF-α,IL-1β,IL-6,TLR4 and NF-κB. However,high dose of rb LBP(20 μg) could obviously attenuate the LPSinduced inflammatory response as indicated by the decreased MPO activity,and by the decreased expression of TNF-α,IL-1β,IL-6,TLR4 and NF-κB. [Conclusions]In summary,rb LBP may play a dual effect in the LPS-induced bovine mastitis in a dose-dependent manner,and one possible mechanism can be attributed to the role of rb LBP in activating TLR4/NF-κB signaling pathway.
[Objectives]This experiment was conducted to study the effect of recombinant bovine lipopolysaccharide(LPS)-binding protein(rb LBP) on LPS-induced bovine mastitis and to explore its relevant mechanism whether associated with the activation of TLR4/NF-κB signaling pathway. [Methods]Four lactating Holstein cows(500±25) kg were used in a 4× 4 latin square design and they were divided into the control group,LPS model group infused with LPS and the rb LBP treatment group infused with LPS and different doses of rb LBP concurrently. The experiment was conducted in four phases(15 days per phase). [Results]The results showed that inflammation was induced in mammary gland after infusing with LPS,and compared with the LPS model group,low dose of LBP(1 μg) enhanced the inflammatory response in mammary gland as indicated by the increased MPO activity and by the increased expression of TNF-α,IL-1β,IL-6,TLR4 and NF-κB. However,high dose of rb LBP(20 μg) could obviously attenuate the LPSinduced inflammatory response as indicated by the decreased MPO activity,and by the decreased expression of TNF-α,IL-1β,IL-6,TLR4 and NF-κB. [Conclusions]In summary,rb LBP may play a dual effect in the LPS-induced bovine mastitis in a dose-dependent manner,and one possible mechanism can be attributed to the role of rb LBP in activating TLR4/NF-κB signaling pathway.
引文
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[16]Cronin J G,Turner M L,Goetze L,et al.Toll-like receptor 4 and MYD88-dependent signaling mechanisms of the innate immune system are essential for the response to lipopolysaccharide by epithelial and stromal cells of the bovine endometrium[J].Biol Reprod,2012,86(2):51.
[17]Liu M,Song S,Li H,et al.The protective effect of caffeic acid against inflammation injury of primary bovine mammary epithelial cells induced by lipopolysaccharide[J].J Dairy Sci,2014,97(5):2856-2865.
[18]Brightbill H D,Mondlin R L.Toll-like receptors:molecular mechanisms of the mammalian immune response[J].Immunology,2000,101(1):1-10.
[19]Ghosh S,Febin J.Non-canonical pathway network modelling and ubiquitination site prediction through homology modelling of NF-kappa B[J].Gene,2016,581(1):48-56.
[20]Wurfel L M M,Kunitake S T,Lichenatein H,et al.Lipopolysaccharide(LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS[J].J Exp Med,1994,180(3):1025-1035.
[2]王亨,孟霞,邱昌伟,等.脂多糖诱导奶牛乳腺上皮细胞先天性免疫反应[J].中国兽医学报,2010,30(3):398-401.Wang H,Meng X,Qiu C W,et al.The innate immune responses of bovine mammary epithelial cells stimulated by lipopolysaccharide[J].Chinese Journal of Veterinary Science,2010,30(3):398-401(in Chinese with English abstract).
[3]Wang Y,Zarlenga D S,Paape M J,et al.Recombinant bovine soluble CD14 sensitizes the mammary gland to lipopolysaccharide[J].Vet Immunol Immunopathol,2002,86(1/2):115-124.
[4]Huang X,Zeng Y,Jiang Y,et al.Lipopolysaccharide-binding protein downregulates fractalkine through activation of p38 MAPK and NF-kappa B[J].Mediators Inflamm,2017.DOI:10.1155/2017/9734837.
[5]余盼,孙宇,王根林,等.乳房炎奶牛乳腺组织中TLR4/p38的表达差异分析[J].南京农业大学学报,2015,38(6):967-972.DOI:10.7685/j.issn.1000-2030.2015.06.014.Yu P,Sun Y,Wang G L,et al.Alteredexpressions of TLR4/p38 singaling pathway in mammary tissue of Chinese Holstein cows with mastitis[J].Journal of Nanjing Agricultural University,2015,38(6):967-972(in Chinese with English abstract).
[6]Strandberg Y,Grgay C,Vuocolo T,et al.Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells[J].Cytokine,2005,31(1):72-86.
[7]Reyad Y A,Berbar M A,Hussain M.Comparison of statistical,LBP,and multi-resolution analysis features for breast mass classification[J].J Med Syst,2014,38(9):100.
[8]Gilbert F B,Cunha P,Jensen K et al.Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system[J].Vet Res,2013,44:40.
[9]Sun Y,Li L,Wu J,et al.Bovine recombinant lipopolysaccharide binding protein(BRLBP)regulated apoptosis and inflammation response in lipopolysaccharide-challenged bovine mammary epithelial cells(BMEC)[J].Mol Immunol,2015,65(2):205-214.
[10]Reid E D,Fried K,Velasco J M,et al.Correlation of rectal temperature and peripheral temperature from implantable radio-frequency microchips in Holstein steers challenged with lipopolysaccharide under thermoneutral and high ambient temperatures[J].J Dairy Sci,2012,90(13):4788-4794.
[11]Zarrin M,Wellnttz O,van Dorland H A,et al.Induced hyperketonemia affects the mammary immune response during lipopolysaccharide challenge in dairy cows[J].J Dairy Sci,2014,97(1):330-339.
[12]甘宗辉,杨章平,李云龙,等.奶牛乳房炎的细菌感染与奶中体细胞数及乳成分的关系[J].畜牧兽医学报,2013,44(6):972-979.Gan Z H,Yang Z P,Li Y L,et al.Relationship of bacterial infection with somatic cell count and milk composition in dairy cows with mastitis[J].Acta Veterinara et Zootechnica Sinica,2013,44(6):972-979(in Chinese with English abstract).
[13]汪悦,王炳,苏汉书,等.牛奶体细胞生成与乳品质量和安全的关系[J].动物营养学报,2017,29(7):2269-2277.Wang Y,Wang B,Su H S,et al.Relationship between milk somatic cell formation and milk quality and safety[J].Chinese Journal of Animal Nutrition,2017,29(7):2269-2277(in Chinese with English abstract).
[14]Vels L,Rontved C M,Bjerring M,et al.Cytokine and acute phase protein gene expression in repeated liver biopsies of dairy cows with a lipopolysaccharide-induced mastitis[J].J Dairy Sci,2009,92(3):922-934.
[15]Zhang Z,Wang X,Li R,et al.Genetic mutations potentially cause two novel NCF1 splice variants up-regulated in the mammary gland,blood and neutrophil of cows infected by Escherichia coli[J].Microbiol Res,2015,174:24-32.
[16]Cronin J G,Turner M L,Goetze L,et al.Toll-like receptor 4 and MYD88-dependent signaling mechanisms of the innate immune system are essential for the response to lipopolysaccharide by epithelial and stromal cells of the bovine endometrium[J].Biol Reprod,2012,86(2):51.
[17]Liu M,Song S,Li H,et al.The protective effect of caffeic acid against inflammation injury of primary bovine mammary epithelial cells induced by lipopolysaccharide[J].J Dairy Sci,2014,97(5):2856-2865.
[18]Brightbill H D,Mondlin R L.Toll-like receptors:molecular mechanisms of the mammalian immune response[J].Immunology,2000,101(1):1-10.
[19]Ghosh S,Febin J.Non-canonical pathway network modelling and ubiquitination site prediction through homology modelling of NF-kappa B[J].Gene,2016,581(1):48-56.
[20]Wurfel L M M,Kunitake S T,Lichenatein H,et al.Lipopolysaccharide(LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS[J].J Exp Med,1994,180(3):1025-1035.