牛磺酸对乳房链球菌感染引起的氧化应激影响及其机制
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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.
[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.
引文
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[14] 代斌,卢劲晔,郑六海,等.牛磺酸对乳房链球菌侵染乳腺上皮细胞所致炎性变化的缓解作用[J].畜牧与兽医,2015,47(7):25-30.Dai B,Lu J Y,Zheng L H,et al.Attenuation of taurine on Streptococcus uberis-induced inflammation changes in mouse mammary epithelial cells[J].Animal Husbandry & Veterinary Medicine,2015,47(7):25-30(in chinese with English abstract).
[15] Harasym J,Oledzki R.Effect of fruit and vegetable antioxidants on total antioxidant capacity of blood plasma[J].Nutrition,2014,30(5):511-517.
[16] 韩月,黄友解,王友明.氧化应激对动物肠道的危害及其营养学缓解措施[J].上海畜牧兽医通讯,2017(4):44-49.Han Y,Huang Y J,Wang Y M.Damage of oxidative stress to animal intestine and its nutritional mitigation measures[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2017(4):44-49(in chinese with English abstract).
[17] Kwiecien S,Jasnos K,Magierowski M,et al.Lipid peroxidation,reactive oxygen species and antioxidative factors in the pathogenesis of gastric mucosal lesions and mechanism of protection against oxidative stress-induced gastric injury[J].J Physiol Pharmacol,2014,65(5):613-622.
[18] Angelova P R,Horrocks M H,Klenerman D,et al.Lipid peroxidation is essential for α-synuclein-induced cell death[J].J Neurochem,2015,133(4):582-589.
[19] Meng S L,Chen J Z,Xu P,et al.Hepatic antioxidant enzymes SOD and CAT of Nile tilapia(Oreochromis niloticus)in response to pesticide methomyl and recovery pattern[J].Bull Environ Contam Toxicol,2014,92(4):388-392.
[20] Ding X,Wang D,Li L L,et al.Dehydroepiandrosterone ameliorates H2O2-induced Leydig cells oxidation damage and apoptosis through inhibition of ROS production and activation of PI3K/Akt pathways[J].The International Journal of Biochemistry & Cell Biology,2016,70:126-139.
[21] Song Y,Huang L L,Yu J F.Effects of blueberry anthocyanins on retinal oxidative stress and inflammation in diabetes through Nrf2/HO-1 signaling[J].Journal of Neuroimmunology,2016,301:1-6.
[22] 孔媛.牛磺酸保护三氧化二砷引起的子代大鼠胰腺自噬性损伤的实验研究[D].大连:大连医科大学,2017.Kong Y.Investigation of taurine’s protection against As2O3-induced autophagy in livers of rats’ offspring[D].Dalian:Dalian Medical University,2017(in chinese with English abstract).
[23] 李乐慧,徐兆发,奉姝,等.牛磺酸对甲基汞染毒大鼠脑皮质Nrf2及HO-1、γ-GCS、Gpx-1表达的影响[J].中国工业医学杂志,2013,26(3):163-166.Li L H,Xu Z F,Feng Z,et al.Effect of taurine on expression of Nrf2,HO-1,γ-GCS and Gpx-1 in cerebral cortex of methylmercury exposed rat[J].Chinese J Ind Med,2013,26(3):163-166(in chinese with English abstract).
[24] Nielsen C U,Bjerg M,Ulaganathan N,et al.Oral and intravenous pharmacokinetics of taurine in sprague-dawley rats:the influence of dose and the possible involvement of the proton-coupled amino acid transporter,PAT1,in oral taurine absorption[J].Physiol Rep,2017,5(19):e13467.
[25] Anderson C M H,Howard A,Walters J R F,et al.Taurine uptake across the human intestinal brush-border membrane is via two transporters:H+-coupled PAT1(SLC36A1)and Na+-and Cl--dependent TauT(SLC6A6)[J].J Physiol,2009,587:731-744.
[2] Nair N,Gongora E.Oxidative stress and cardiovascular aging:interaction between NRF-2 and ADMA[J].Current Cardiology Reviews,2017,13:183-188.
[3] Kattoor A J,Pothineni N V K,Palagiri D,et al.Oxidative stress in atherosclerosis[J].Curr Atheroscler Rep,2017,19(11):42.
[4] Warolin J,Coenen K R,Kantor J L,et al.The relationship of oxidative stress,adiposity and metabolic risk factors in healthy black and white american youth[J].Pediatr Obes,2014,9(1):43-52.
[5] Morais J B S,Severo J S,Santos L R,et al.Role of magnesium in oxidative stress in individuals with obesity[J].Biol Trace Elem Res,2017,176(1):20-26.
[6] Hayes J D,Dinkova-Kostova A T.The Nrf2 regulatory network provides an interface between redox and intermediary metabolism[J].Trends Biochem Sci,2014,39(4):199-218.
[7] Ma Q.Role of Nrf2 in oxidative stress and toxicity[J].Annu Rev Pharmacol Toxico,2013,53:401-426.
[8] Jaiswal A K.Nrf2 signaling in coordinated activation of antioxidant gene expression[J].Free Radic Biol Med,2004,36(10):1199-1207.
[9] Suzuki T,Motohashi H,Yamamoto M.Toward clinical application of the Keap1-Nrf2 pathway[J].Trends Pharmacol Sci,2013,34(6):340-346.
[10] Baird L,Llères D,Swift S,et al.Regulatory flexibility in the Nrf2-mediated stress response is conferred by conformational cycling of the Keap1-Nrf2 protein complex[J].Proc Natl Acad Sci USA,2013,110:15259-15264.
[11] Sihvola V,Levonen A L.Keap1 as the redox sensor of the antioxidant response[J].Arch Biochem Biophys,2017,617:94-100.
[12] Canning P,Cooper C D O,Krojer T,et al.Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases[J].Biol Chem,2013,288(11):7803-7814.
[13] Zheng L H,Xu Y Y,Lu J Y,et al.Variant innate immune responses of mammary epithelial cells to challenge by Staphylococcus aureus,Escherichia coli and the regulating effect of taurine on these bioprocesses[J].Free Radic Biol Med,2016,96:166-180.
[14] 代斌,卢劲晔,郑六海,等.牛磺酸对乳房链球菌侵染乳腺上皮细胞所致炎性变化的缓解作用[J].畜牧与兽医,2015,47(7):25-30.Dai B,Lu J Y,Zheng L H,et al.Attenuation of taurine on Streptococcus uberis-induced inflammation changes in mouse mammary epithelial cells[J].Animal Husbandry & Veterinary Medicine,2015,47(7):25-30(in chinese with English abstract).
[15] Harasym J,Oledzki R.Effect of fruit and vegetable antioxidants on total antioxidant capacity of blood plasma[J].Nutrition,2014,30(5):511-517.
[16] 韩月,黄友解,王友明.氧化应激对动物肠道的危害及其营养学缓解措施[J].上海畜牧兽医通讯,2017(4):44-49.Han Y,Huang Y J,Wang Y M.Damage of oxidative stress to animal intestine and its nutritional mitigation measures[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2017(4):44-49(in chinese with English abstract).
[17] Kwiecien S,Jasnos K,Magierowski M,et al.Lipid peroxidation,reactive oxygen species and antioxidative factors in the pathogenesis of gastric mucosal lesions and mechanism of protection against oxidative stress-induced gastric injury[J].J Physiol Pharmacol,2014,65(5):613-622.
[18] Angelova P R,Horrocks M H,Klenerman D,et al.Lipid peroxidation is essential for α-synuclein-induced cell death[J].J Neurochem,2015,133(4):582-589.
[19] Meng S L,Chen J Z,Xu P,et al.Hepatic antioxidant enzymes SOD and CAT of Nile tilapia(Oreochromis niloticus)in response to pesticide methomyl and recovery pattern[J].Bull Environ Contam Toxicol,2014,92(4):388-392.
[20] Ding X,Wang D,Li L L,et al.Dehydroepiandrosterone ameliorates H2O2-induced Leydig cells oxidation damage and apoptosis through inhibition of ROS production and activation of PI3K/Akt pathways[J].The International Journal of Biochemistry & Cell Biology,2016,70:126-139.
[21] Song Y,Huang L L,Yu J F.Effects of blueberry anthocyanins on retinal oxidative stress and inflammation in diabetes through Nrf2/HO-1 signaling[J].Journal of Neuroimmunology,2016,301:1-6.
[22] 孔媛.牛磺酸保护三氧化二砷引起的子代大鼠胰腺自噬性损伤的实验研究[D].大连:大连医科大学,2017.Kong Y.Investigation of taurine’s protection against As2O3-induced autophagy in livers of rats’ offspring[D].Dalian:Dalian Medical University,2017(in chinese with English abstract).
[23] 李乐慧,徐兆发,奉姝,等.牛磺酸对甲基汞染毒大鼠脑皮质Nrf2及HO-1、γ-GCS、Gpx-1表达的影响[J].中国工业医学杂志,2013,26(3):163-166.Li L H,Xu Z F,Feng Z,et al.Effect of taurine on expression of Nrf2,HO-1,γ-GCS and Gpx-1 in cerebral cortex of methylmercury exposed rat[J].Chinese J Ind Med,2013,26(3):163-166(in chinese with English abstract).
[24] Nielsen C U,Bjerg M,Ulaganathan N,et al.Oral and intravenous pharmacokinetics of taurine in sprague-dawley rats:the influence of dose and the possible involvement of the proton-coupled amino acid transporter,PAT1,in oral taurine absorption[J].Physiol Rep,2017,5(19):e13467.
[25] Anderson C M H,Howard A,Walters J R F,et al.Taurine uptake across the human intestinal brush-border membrane is via two transporters:H+-coupled PAT1(SLC36A1)and Na+-and Cl--dependent TauT(SLC6A6)[J].J Physiol,2009,587:731-744.