摘要
[目的]本文旨在探究牛磺酸对乳房链球菌(Streptococcus uberis)感染引起的氧化应激反应的影响及机制。[方法]用70 mmol·L^(-1)牛磺酸(taurine)处理牛乳腺上皮细胞(MAC-T)4 h,加入S.uberis[感染复数(MOI)=10]继续培养3 h,收集细胞;用靶向siRNA干扰牛磺酸的2个转运载体(TauT、PAT1)的表达,再用70 mmol·L^(-1)牛磺酸处理4 h,收集样品。通过Western blot、流式细胞术、试剂盒(ABTS法、TBA法、WST-1法、钼酸铵法等)、免疫荧光等方法,检测Keap1和Nrf2蛋白的表达,活性氧(ROS)和细胞内总抗氧化能力(T-AOC)水平,丙二醛(MDA)含量,超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性及Nrf2蛋白在细胞中的定位。[结果]S.uberis感染能够显著降低T-AOC水平及提高MDA和ROS含量,使SOD和CAT活性显著提高(P<0.05)。与S.uberis感染组相比,牛磺酸预处理后感染细菌显著提高胞内T-AOC水平及SOD和CAT活性,降低MDA和ROS含量,并抑制Keap1的表达,促进Nrf2的表达及入核(P<0.05);抑制牛磺酸转运载体TauT、PAT1的表达后,Keap1的降低及Nrf2的升高被显著抑制(P<0.05)。[结论]牛磺酸可能通过转运载体TauT、PAT1进入乳腺上皮细胞,并通过激活Keap1-Nrf2信号通路缓解S.uberis感染引起的氧化应激反应。
[Objectives]The paper aims to investigate the effects of taurine on oxidative stress induced by Streptococcus uberis and its mechanism.[Methods]In this study,bovine mammary epithelial cells(MAC-T)was treated with 70 mmol·L^-1 taurine for 4 h,then the S.uberis[multiplicity of infection(MOI)=10]was added,and after incubation for 3 h,the cells were collected.Then the targeted siRNA was used to interfere with the genes expression of two taurine transporters(TauT,PAT1).After that,the bovine mammary epithelial cells(MAC-T)were treated with 70 mmol·L^-1 taurine for 4 h and the samples ware collected.The contents of key proteins in Keap1-Nrf2 signaling pathway,reactive oxygen species(ROS),total antioxidant capacity(T-AOC),malondialdehyde(MDA),and the activities of superoxide dismutase(SOD),catalase(CAT)and localization of Nrf2 protein were determined by the the methods of Western blot,flow cytometry and biochemical kits(ABTS,TBA,WST-1,ammonium molybdate,et al methods)and immunofluorescence.[Results]The results indicated that the cells infected with S.uberis could significantly reduce T-AOC level,increase the contents of MDA and ROS,and promote the activities of SOD and CAT(P<0.05).Compared with the S.uberis group,the cells treated with taurine before infecting with S.uberis could significantly increase the content of T-AOC,activities of SOD,CAT and reduce the contents of the MDA and ROS(P<0.05).It was also found that the cells treated with taurine before infecting with S.uberis could significantly inhibite the expression of Keap1,but promote the expression and nucleation of Nrf2(P<0.05).After inhibiting the genes expression of TauT and PAT1,the decrease of Keap1 and the increase of Nrf2 were significantly inhibited(P<0.05).[Conclusions]Taurine could enter into mammary epithelial cells through the transport vectors TauT and PAT1,and it could activate the Keap1-Nrf2 pathway to alleviate the oxidative stress caused by S.uberis infection.
作者
王正磊
周艺琳
周美意
董心仪
臧琦铭
PHOUTHAPANE Vanhnaseng
苗晋锋
WANG Zhenglei;ZHOU Yilin;ZHOU Meiyi;DONG Xinyi;ZANG Qiming;MIAO Jinfeng(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Animal Science Center at Biotechnology and Ecology Institute,Ministry of Science and Technology of Laos,Vientiane 01000,Laos)
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2019年第3期491-498,共8页
Journal of Nanjing Agricultural University
基金
国家重点研发计划项目(2017YFD0501000)
国家自然科学基金项目(31672515)
