乳头灌注脂多糖对奶牛乳腺组织及机体免疫的影响
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摘要
旨在通过乳头灌注脂多糖(LPS)体外模拟奶牛急性乳腺炎,研究奶牛乳腺组织及机体免疫活化状态。选用4头泌乳后期的荷斯坦奶牛16个乳区为研究对象,采用交叉试验设计,每期试验2 d,试验间隔10 d,试验组乳头管乳头灌注LPS 0. 1 mg,对照组乳头管灌注等量生理盐水。结果:试验组灌注LPS后,奶牛体温值和乳汁中体细胞数随时间变化而逐渐升高,而奶牛血液中白细胞数随时间变化而逐渐降低;乳腺组织的腺泡腔内充斥大量炎性细胞,部分腺泡组织萎缩甚至崩塌;试验组灌注LPS 12 h后奶牛血清中的髓过氧化物(MPO)活性极显著下降(P<0. 01),而乳腺组织中的MPO活性则极显著上升(P<0. 01);奶牛血清及乳清中炎症因子肿瘤坏死因子(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)的蛋白含量明显上升(P<0. 01);乳腺组织中Toll样受体4 (TLR4)、核因子NF-κB (p65)蛋白的表达量相比对照组极显著上升(P<0. 01)。研究表明,LPS能够诱导奶牛发生乳腺炎,使乳腺组织发生损伤,并激活机体的TLR4/NF-κB信号通路进而促进其下游炎症因子TNF-α、IL-1β、IL-6的基因的表达,使机体发生免疫应答。
This study was to investigate the immune effect of mammary tissue and the body of cows with mastitis induced by LPS. Four latelactating Holstein cows were divided into two groups randomly. Different treatment methods were adopted for different groups: the trial group were injected with 0. 1 mg LPS,and the control group with sterile saline. According to the orthogonal design method,the experiment was performed twice repeatedly,each lasting for two days at an interval of ten days. The results showed that the number of somatic cells and rectal temperature increased significantly in the trial group. The values reached at peak value at 9 hour of the experiment,and then they began to gradually decrease. On the contrary,the white blood cells of the trial group dropped significantly compared with the control group; the value reached its nadir at 9 hour of the experiment,then it began to gradually increase. The histopathological test showed that the mammary tissues were infiltrated with a number of inflammatory cells 12 hours after injection with LPS. Moreover,the gland bubble was in the state of atrophy,and the alveolar form was significantly damaged. The MPO activity in the mammary gland increased obviously 12 hours after injection with LPS (P<0. 01). However,the changes in the MPO activity in the serum showed an entirely opposite trend (P<0. 01). The concentrations of IL-1β,IL-6,and TNF-αsignificantly increased 12 hours after LPS-stimulation,even in the serum and mammary gland tissue homogenates (P<0. 01). Stimulation with LPS improved the protein levels of TLR4 and NF-κB in the mammary gland (P<0. 01). In conclusion,the model of cow mastitis induced by LPS was built up here,and the mammary tissues were damaged. LPS might play a positive effect by regulating the production of TNF-α,IL-1β and IL-6 via the TLR-4/NF-κB signaling pathway in the LPS-induced cow mastitis,and finally,the body's immune response was induced.
This study was to investigate the immune effect of mammary tissue and the body of cows with mastitis induced by LPS. Four latelactating Holstein cows were divided into two groups randomly. Different treatment methods were adopted for different groups: the trial group were injected with 0. 1 mg LPS,and the control group with sterile saline. According to the orthogonal design method,the experiment was performed twice repeatedly,each lasting for two days at an interval of ten days. The results showed that the number of somatic cells and rectal temperature increased significantly in the trial group. The values reached at peak value at 9 hour of the experiment,and then they began to gradually decrease. On the contrary,the white blood cells of the trial group dropped significantly compared with the control group; the value reached its nadir at 9 hour of the experiment,then it began to gradually increase. The histopathological test showed that the mammary tissues were infiltrated with a number of inflammatory cells 12 hours after injection with LPS. Moreover,the gland bubble was in the state of atrophy,and the alveolar form was significantly damaged. The MPO activity in the mammary gland increased obviously 12 hours after injection with LPS (P<0. 01). However,the changes in the MPO activity in the serum showed an entirely opposite trend (P<0. 01). The concentrations of IL-1β,IL-6,and TNF-αsignificantly increased 12 hours after LPS-stimulation,even in the serum and mammary gland tissue homogenates (P<0. 01). Stimulation with LPS improved the protein levels of TLR4 and NF-κB in the mammary gland (P<0. 01). In conclusion,the model of cow mastitis induced by LPS was built up here,and the mammary tissues were damaged. LPS might play a positive effect by regulating the production of TNF-α,IL-1β and IL-6 via the TLR-4/NF-κB signaling pathway in the LPS-induced cow mastitis,and finally,the body's immune response was induced.
引文
[1]徐伟,陈亚妮,何珊珊,等.奶牛急性大肠杆菌性乳房炎病例[J].中国兽医杂志,2016,52(09):119-120.
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[4]Kalmus P,Aasm?e B,K?rssin A,et al.Udder pathogens and their resistance to antimicrobial agents in dairy cows in Estonia[J].Acta Vet Scand,2011,53.
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[6]Paape M J,Lilius E M,Wiitanen P A,et al.Intramammary defense against infections induced by Escherichia coli in cows[J].Am J Vet Res,1996,57:477-482.
[7]Gonen E,Vallon-Eberhard A,Elazar S,et al.Toll-like receptor 4is needed to restrict the invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine model of acute mastitis[J].Cell Microbiol,2007,9(12):2826-2838.
[8]Takeuchi O,Hoshino K,Akira S,et al.TLR2-deficient and My D88-deficient mice are highly susceptible to Staphylococcus aureus infection[J].J Immunol,2000,165(10):5392-5396.
[9]Brandenburg K,Heinbockel L,Correa W,et al.Supramolecular structure of enterobacterial wild-type lipopolysaccharides (LPS),fractions thereof,and their neutralization by Pep[J].J Struct Biol,2016,194:68-77.
[10]袁晓,范永升,谢冠群.基于“TLR4/NF-kB”信号通路研究“加味四妙丸”治疗急性痛风性关节炎大鼠的作用机制[J].浙江中医药大学学报,2017(1):17-24.
[11]Schmitz S,Pfaffl M W,Meyer H,et al.Short-term changes of mR-NA expression of various inflammatory factors and milk proteins in mammary tissue during LPS-induced mastitis.[J].Domest Anim Endocrin,2004,26:111-126.
[12]Wellnitz O,Baumert A,Saudenowa M,et al.Immune response of bovine milk somatic cells to endotoxin in healthy quarters with normal and very low cell counts[J].J Dairy Res,2010,77:452-459.
[13]Morimoto K,Kanda N,Shinde S,et al.Effect of enterotoxigenic Escherichia coli vaccine on innate immune function of bovine mammary gland infused with lipopolysaccharide[J].J Dairy Sci,2012,95:5067-5074.
[14]Wellnitz O,Kerr D E.Cryopreserved bovine mammary cells to model epithelial response to infection[J].Vet Immunol Immunop,2004,101:191-202.
[15]Strandberg Y,Gray C,Vuocolo T,et al.Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells[J].Cytokine,2005,31:72-86.
[16]刘佳佳,文佳军,余雄.不同音乐对奶牛产奶性能影响的研究[J].黑龙江畜牧兽医,2015(3):28-33.
[17]Zhang H,Issekutz A C.Down-modulation of monocyte transendothelial migration and endothelial adhesion molecule expression by fibroblast growth factor:reversal by the anti-angiogenicagent SU6668[J].Am J Pathol,2002,160(6):2219-2230.
[18]Sordillo L M,Babiuk L A.Modulation of bovine mammary neutrophil function during the periparturient period following in vitro exposure to recombinant bovine interferon gamma[J].Vet Immunol Immunopathol,1991,27(4):393-402.
[19]Vels L,Rontved C M,Bjerring M,et al.Cytokine and individual variability of the lipopolysaccharide-induced bovine acute phase protein response[J].J Dairy Sci,2004,87:3330-3339.
[20]Bannerman D D,Paape M J,Lee J W,et al.Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection[J].Clin Diagn Lab Immunol,2004,11(3):463-472.
[21]Takeuchi O,Hoshino K,Akira S.et al.TLR2-deficient and My D88-deficient mice are highly susceptible to Staphylococcus aureus infection[J].J Immunol,2000,165(10):5392-5396.
[22]Beutler B.Inferencesquestions and possibilities in Toll-like receptor signaling[J].Nature,2004,430(6996):257-263.
[2]Burvenich C,Van Merris V,Mehrzad J,et al.Severity of E.coli mastitis is mainly determined by cow factors[J].Vet Res,2003,34:521-564.
[3]Bradley A J,Leach K A,Breen J E,et al.Survey of the incidence and aetiology of mastitis on dairy farms in England and Wales[J].Vet Rec.2007,160:253-258.
[4]Kalmus P,Aasm?e B,K?rssin A,et al.Udder pathogens and their resistance to antimicrobial agents in dairy cows in Estonia[J].Acta Vet Scand,2011,53.
[5]Lee J,Paape M J,Elsasser T H,et al.Elevated milk soluble CD14in bovine mammary glands challenged with Escherichia coli lipopolysaccharide[J].J Dairy Sci,2003,86:2382-2389.
[6]Paape M J,Lilius E M,Wiitanen P A,et al.Intramammary defense against infections induced by Escherichia coli in cows[J].Am J Vet Res,1996,57:477-482.
[7]Gonen E,Vallon-Eberhard A,Elazar S,et al.Toll-like receptor 4is needed to restrict the invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine model of acute mastitis[J].Cell Microbiol,2007,9(12):2826-2838.
[8]Takeuchi O,Hoshino K,Akira S,et al.TLR2-deficient and My D88-deficient mice are highly susceptible to Staphylococcus aureus infection[J].J Immunol,2000,165(10):5392-5396.
[9]Brandenburg K,Heinbockel L,Correa W,et al.Supramolecular structure of enterobacterial wild-type lipopolysaccharides (LPS),fractions thereof,and their neutralization by Pep[J].J Struct Biol,2016,194:68-77.
[10]袁晓,范永升,谢冠群.基于“TLR4/NF-kB”信号通路研究“加味四妙丸”治疗急性痛风性关节炎大鼠的作用机制[J].浙江中医药大学学报,2017(1):17-24.
[11]Schmitz S,Pfaffl M W,Meyer H,et al.Short-term changes of mR-NA expression of various inflammatory factors and milk proteins in mammary tissue during LPS-induced mastitis.[J].Domest Anim Endocrin,2004,26:111-126.
[12]Wellnitz O,Baumert A,Saudenowa M,et al.Immune response of bovine milk somatic cells to endotoxin in healthy quarters with normal and very low cell counts[J].J Dairy Res,2010,77:452-459.
[13]Morimoto K,Kanda N,Shinde S,et al.Effect of enterotoxigenic Escherichia coli vaccine on innate immune function of bovine mammary gland infused with lipopolysaccharide[J].J Dairy Sci,2012,95:5067-5074.
[14]Wellnitz O,Kerr D E.Cryopreserved bovine mammary cells to model epithelial response to infection[J].Vet Immunol Immunop,2004,101:191-202.
[15]Strandberg Y,Gray C,Vuocolo T,et al.Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells[J].Cytokine,2005,31:72-86.
[16]刘佳佳,文佳军,余雄.不同音乐对奶牛产奶性能影响的研究[J].黑龙江畜牧兽医,2015(3):28-33.
[17]Zhang H,Issekutz A C.Down-modulation of monocyte transendothelial migration and endothelial adhesion molecule expression by fibroblast growth factor:reversal by the anti-angiogenicagent SU6668[J].Am J Pathol,2002,160(6):2219-2230.
[18]Sordillo L M,Babiuk L A.Modulation of bovine mammary neutrophil function during the periparturient period following in vitro exposure to recombinant bovine interferon gamma[J].Vet Immunol Immunopathol,1991,27(4):393-402.
[19]Vels L,Rontved C M,Bjerring M,et al.Cytokine and individual variability of the lipopolysaccharide-induced bovine acute phase protein response[J].J Dairy Sci,2004,87:3330-3339.
[20]Bannerman D D,Paape M J,Lee J W,et al.Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection[J].Clin Diagn Lab Immunol,2004,11(3):463-472.
[21]Takeuchi O,Hoshino K,Akira S.et al.TLR2-deficient and My D88-deficient mice are highly susceptible to Staphylococcus aureus infection[J].J Immunol,2000,165(10):5392-5396.
[22]Beutler B.Inferencesquestions and possibilities in Toll-like receptor signaling[J].Nature,2004,430(6996):257-263.