动物营养学报2019，Vol.31Issue(8)：3706-3718,13.DOI:10.3969/j.issn.1006⁃267x.2019.08.032
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
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
摘要
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 concentra?tion, 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 apopto?sis 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 ac?tivator 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, pro?mote 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.[ Chinese Journal of Animal Nutrition, 2019, 31( 8) :3706?3718]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Key words
Streptococcus agalactiae/bovine mammary gland epithelial cells/milk protein synthesis/signa⁃ling pathways分类
农业科技引用本文复制引用
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):3706-3718,13.基金项目
"十三五"国家重大科技专项(2016YFD0700201,2016YFD0700205,2017YFD0701604) （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) （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) （ SQKM201710020011）
北京市博士后基金 （）
