乳房链球菌感染对乳腺上皮细胞中氨基酸代谢的影响  

The Effect of Streptococcus uberis Infection on Amino Acid Metabolism in Mammary Epithelial Cells

作　　者：卢劲晔[1] 高亚兵 韩心茹 刘钰臻 赵家玉 LU Jinye;GAO Yabing;HAN Xinru;LIU Yuzhen;ZHAO Jiayu(Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China;Key Laboratory of Animal Physiology and Biochemistry of Ministry of Agriculture,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]江苏农牧科技职业学院,泰州225300 [2]南京农业大学动物医学院/农业部动物生理生化重点开放实验室,南京210095

出　　处：《畜牧兽医学报》2024年第4期1766-1776,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：江苏农牧科技职业学院科研项目(NSF2021ZR05);国家自然科学基金项目(32072867,32273013)。

摘　　要：代谢是一切生理活动的基石,氨基酸代谢作为三大物质代谢之一,参与机体的多种生理活动,与宿主免疫细胞的功能有密切关系,在疾病的发展中具有不可替代的作用。为探究乳房链球菌(Streptococcus uberis,S.uberis)感染对乳腺上皮细胞中氨基酸代谢的影响,本研究以S.uberis和乳腺上皮细胞系(EpH4-Ev)为研究对象,采用靶向氨基酸代谢组学技术对细胞中的氨基酸进行定量分析,并采用RT-qPCR和Western blot方法对关键氨基酸——精氨酸代谢通路关键酶进行验证。结果表明:S.uberis感染细胞后,细胞中甘氨酸、苏氨酸、鸟氨酸、天冬酰胺和谷氨酰胺的含量被显著上调(P<0.05),谷氨酸、脯氨酸、精氨酸、丝氨酸、组氨酸、缬氨酸、异亮氨酸、丙氨酸、酪氨酸、色氨酸、赖氨酸和天冬氨酸的含量被显著下调(P<0.05),其中,精氨酸含量变化极显著(P<0.000 1,VIP>1.2),天冬酰胺和谷氨酰胺两种生糖氨基酸显著上升(P<0.05),赖氨酸和亮氨酸两种生酮氨基酸含量显著下降(P<0.05)。进一步研究精氨酸分解代谢通路发现:S.uberis感染乳腺上皮细胞会显著上调精氨酸代谢关键酶NOS2的mRNA和蛋白表达水平(P<0.05),显著下调ARG1的表达水平(P<0.05)。与对照组相比,S.uberis感染乳腺后,显著上调了细胞中促炎因子IL-1β、TNF-α表达(P<0.05)。综上所述,S.uberis感染造成乳腺上皮细胞氨基酸代谢紊乱,加速细胞内精氨酸耗竭,诱发炎症反应。结果提示,精氨酸在宿主抵抗S.uberis感染过程中具有重要作用,可作为奶牛乳腺炎早期诊断和未来防治的突破口。Metabolism is the cornerstone of all physiological activities,and amino acid metabolism,as one of the three major substance metabolisms,is involved in a variety of physiological activities of the host organism.It is closely related to the function of host immune cells and has an irreplaceable role in the development of diseases.To investigate the effect of Streptococcus uberis(S.uberis)infection on amino acid metabolism in mammary epithelial cells,S.uberis and mammary epithelial cell line(EpH4-Ev)were used for quantitative analysis of amino acids of cells by targeted amino acid metabolomics techniques in this study.The key enzymes expression of key amino acid(arginine)metabolic pathway was also validated by RT-qPCR and Western blot.The results showed that the levels of glycine,threonine,ornithine,asparagine and glutamine were significantly up-regulated(P<0.05),and the levels of glutamate,proline,arginine,serine,histidine,valine,isoleucine,alanine,tyrosine,tryptophan,lysine and aspartate were significantly down-regulated(P<0.05)in S.uberis-infected cells,with arginine content was most significantly changed(P<0.0001,VIP>1.2).Two glycogenic amino acids,asparagine and glutamine,were significantly increased(P<0.05),two ketogenic amino acids,lysine and leucine,were significantly decreased(P<0.05).Further exploration of the arginine catabolic pathway revealed that S.uberis infection of mammary epithelial cells significantly upregulated the mRNA and protein expression levels of NOS2,a key enzyme of arginine metabolism(P<0.05),and significantly downregulated the expression level of ARG1(P<0.05).Compared with the control group,the expression of pro-inflammatory factors IL-1βand TNF-αin the cells challenged with S.uberis were significantly up-regulated(P<0.05),and the expression of anti-inflammatory factor IL-10 was decreased(P<0.05).In summary,S.uberis infection caused disruption of amino acid metabolism in mammary epithelial cells,accelerated intracellular arginine depletion,and initiated inflammatory responses.The resu

关 键 词：乳房链球菌 氨基酸代谢 精氨酸 乳腺炎 

分 类 号：S857.26[农业科学—临床兽医学]