动物营养学报 >

2019 , Vol. 31 >Issue 8: 3706 - 3718

DOI: https://doi.org/10.3969/j.issn.1006-267x.2019.08.032

分子营养 Molecular Nutrition

无乳链球菌通过抑制酪氨酸激酶/信号转导及转录激活因子和哺乳动物雷帕霉素靶蛋白信号通路影响奶牛乳腺上皮细胞乳蛋白的合成

  • 杨德莲 ,
  • 童津津 ,
  • 孙铭维 ,
  • 张婕 ,
  • 张华 ,
  • 熊本海 ,
  • 蒋林树
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  • 1. 奶牛营养学北京市重点实验室, 北京农学院动物科技学院, 北京 102206;
    2. 中国农业科学院北京畜牧兽医研究所, 北京 100193
杨德莲(1993-),女,云南昭通人,硕士研究生,研究方向为反刍动物营养与免疫。E-mail:1121529738@qq.com

收稿日期: 2019-01-23

  网络出版日期: 2019-08-19

基金资助

“十三五”国家重大科技专项（2016YFD0700201，2016YFD0700205，2017YFD0701604）；北京市现代农业产业技术体系奶牛创新团队；国家自然科学基金（31772629，31702302，31802091）；2015年大北农青年教师科研基金；2016年北京农学院青年教师科研基金；2017年北京市教委科技计划项目（SQKM201710020011）；北京市博士后基金

收起

Streptococcus agalactiae Affects Milk Protein Synthesis in Bovine Mammary Epithelial Cells by Inhibiting Tyrosine Kinase/Signal Transduction and Transcriptional Activators and Mammalian Target of Rapamycin Signaling Pathways

  • YANG Delian ,
  • TONG Jinjin ,
  • SUN Mingwei ,
  • ZHANG Jie ,
  • ZHANG Hua ,
  • XIONG Benhai ,
  • JIANG Linshu
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  • 1. Key Laboratory for Dairy Cow Nutrition, College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, China;
    2. Institute of Animal Science and Veterinary, Chinese Academy of Agricultural Science, Beijing 100193, China

Received date: 2019-01-23

  Online published: 2019-08-19

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摘要

本试验旨在研究无乳链球菌（GBS）对奶牛乳腺上皮细胞（BMECs）乳蛋白合成的影响机理。试验用不同浓度GBS[感染复数（MOI）分别为100、50、10]感染BMECs 1、2、4、6、8、12、18、24 h，每个时间点都设立相应的空白对照，采用乳酸脱氢酶（LDH）试剂盒、扫描电镜以及流式细胞术等方法检测GBS对BMECs活性、形态及凋亡的影响；采用反转录－聚合酶链式反应（RT-PCR）和Western blot检测β-酪蛋白及乳蛋白合成相关基因的mRNA和蛋白表达量。结果表明：1）GBS对BMECs的毒性作用具有明显的时间、剂量依赖性，即随着感染时间的延长、细菌浓度的升高，LDH释放量显著或极显著高于对照组（P<0.05或P<0.01）；GBS感染后细胞形态发生显著变化，感染6 h时，细胞结构断裂，感染8 h时，细胞形态结构受到严重破坏；感染2 h时，BMECs发生了显著的凋亡（P<0.05）；感染6 h时，BMECs发生极显著的凋亡（P<0.01）。2）感染6 h时，GBS导致BMECs中β-酪蛋白以及正调控乳蛋白表达基因酪氨酸激酶2（JAK2）、信号转换及转录激活因子5a（STAT5a）、哺乳动物雷帕霉素靶蛋白（mTOR）、丝氨酸/苏氨酸蛋白激酶1（AKT1）、核糖体蛋白S6（sRPS6）、催乳素受体（PRLR）、est结构域转录因子5（ELF5）mRNA表达量极显著下降（P<0.01）；负调控基因真核翻译起始因子4E结合蛋白1（EIF4E-BP1）mRNA表达量极显著上升（P<0.01）；GBS导致BMECs中β-酪蛋白、STAT5a、磷酸化-信号转导及转录激活因子5a（p-STAT5a）、mTOR、磷酸化-哺乳动物雷帕霉素靶蛋白（p-mTOR）、AKT1蛋白表达量极显著下降（P<0.01），而磷酸化-丝氨酸/苏氨酸蛋白激酶1（p-AKT1）蛋白表达完全受到抑制。在感染8 h时，GBS完全抑制了β-酪蛋白、STAT5a、p-STAT5a、mTOR、p-mTOR、AKT1、p-AKT1的蛋白表达。由此可见，GBS能够损伤BMECs的形态结构，促进细胞凋亡，降低细胞活性，从而影响细胞的正常生理功能，此外，GBS抑制BMECs乳蛋白的合成，主要通过抑制JAK/STAT、mTOR信号通路发挥作用。

关键词： 无乳链球菌; 奶牛乳腺上皮细胞; 乳蛋白合成; 信号通路

本文引用格式

杨德莲 , 童津津 , 孙铭维 , 张婕 , 张华 , 熊本海 , 蒋林树 . 无乳链球菌通过抑制酪氨酸激酶/信号转导及转录激活因子和哺乳动物雷帕霉素靶蛋白信号通路影响奶牛乳腺上皮细胞乳蛋白的合成[J]. 动物营养学报, 2019 , 31(8) : 3706 -3718 . DOI: 10.3969/j.issn.1006-267x.2019.08.032

Abstract

The objective of this study was to explore the mechanism of the effects of Streptococcus agalactiae (GBS) on milk protein synthesis in bovine mammary epithelial cells (BMECs). Three different concentrations of GBS (MOI=100, 50 and 10) were used to infect BMECs for 1, 2, 4, 6, 8, 12, 18, and 24 h, and each time point was set up corresponding blank control. Then,the effects of GBS on the activity, morphology and apoptosis of BMECs were determined using lactate dehydrogenase (LDH) kit, scanning electron microscopy and flow cytometry. RT-PCR and Western blot were used to detect the mRNA and protein expression of related genes of β-casein and lactoprotein synthesis. The results showed as follows:1) the toxicity of GBS to BMECs was time-and dose-dependent. With the prolongation of infection time and the increase of bacterial concentration, LDH release amount was significantly or extremely significantly higher than that of the control group (P<0.05 or P<0.01). The cell morphology changed significantly after GBS infection. At 6 h of infection, the cell structure was broken and the cell morphology was severely damaged at 8 h of infection. Moreover, the apoptosis of BMECs was significant at 2 h (P<0.05) and extremely significant at 6 h of infection (P<0.01). 2) When the infecting time lasted for 6 h, GBS resulted that the mRNA expression levels of β-casein and the genes regulating lactoprotein expression like tyrosine kinase 2 (JAK2), signal transduction and transcription activator 5a (STAT5a), mammalian target of rapamycin (mTOR), serine/threonine protein kinase 1 (AKT1), ribosomal protein S6 (sRPS6), prolactin receptor (PRLR) and est domain transcription factor 5 (ELF5) in BMECs, were extremely significant down-regulated (P<0.01), while the mRNA expression level of negative regulatory gene eukaryotic translation initiation factor 4E binding protein 1 (EIF4E-BP1) was remarkably up-regulated (P<0.01). The protein expression levels of β-casein, STAT5a, p-STAT5a, mTOR, p-mTOR, and AKT1 in BMECs were dramatically down-regulated by GBS (P<0.01), while P-AKT1 protein expression was completely inhibited. At 8 h of infection, GBS completely inhibited the protein expression of β-casein, STAT5a, p-STAT5a. It can be concluded that GBS can damage the morphological structure of BMECs, promote apoptosis and decrease cell viability, at last having an adverse impact on the normal cell physiological functions. In addition, GBS inhibits the lactoprotein synthesis in BMECs mainly by inhibiting the function of the JAK/STAT and mTOR signaling pathways.

Key words： Streptococcus agalactiae; bovine mammary gland epithelial cells; milk protein synthesis; signaling pathways

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