ZEA对小鼠T淋巴细胞活化过程中超微结构及核因子NFAT和NF-κB信号通路的影响
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摘要
为揭示玉米赤霉烯酮(ZEA)对动物免疫功能影响的机制,以免疫磁珠负性分选法从小鼠脾脏中分离得到的高纯度T淋巴细胞为材料,刀豆蛋白A(Con A)作为T淋巴细胞活化刺激剂,研究ZEA对小鼠T淋巴细胞活化过程中细胞超微结构的影响及对核因子NFAT、NF-κB信号通路的影响。试验设空白对照组(不含Con A)、Con A (5μg·mL~(-1))对照组、ZEA(10、20及40μmol·L~(-1))+Con A (5μg·mL~(-1))染毒组,细胞染毒48h后用透射电镜观察细胞结构变化情况。细胞染毒处理24和36h后分别提取细胞浆、细胞核蛋白,Western blot检测NFAT及NF-κB信号通路中关键蛋白表达情况。结果表明:与空白对照组相比,Con A组T淋巴细胞胞浆内NFAT、NF-κB等相关蛋白含量显著或极显著升高;细胞体积增大,细胞浆丰富,核糖体和线粒体明显增多。与Con A组相比,ZEA染毒组T淋巴细胞胞浆内NFAT、NF-κB相关蛋白含量显著或极显著下降,并呈剂量-效应关系;细胞胞浆减少,细胞质出现不同程度的空泡化,线粒体肿胀,核糖体消失,异染色质增多。与空白对照组相比,Con A组T淋巴细胞核内NFAT、NF-κB含量均极显著升高;与Con A组相比,ZEA染毒组T淋巴细胞核内NFAT、NF-κB含量显著或极显著下降,并呈剂量-效应关系。这一研究提示ZEA可造成T淋巴细胞活化过程中细胞超微结构的损伤,抑制小鼠T淋巴细胞活化过程中核因子NFAT、NF-κB信号通路活性,抑制相关蛋白合成,造成T淋巴细胞的活化作用被抑制。
To reveal the mechanism of zearalenone(ZEA)on immune function in mice,the primary spleen lymphocytes stimulated by Concanavalin A(Con A)were used as the experiment material.The effect of ZEA on the ultrastructure and NFAT,NF-κB signaling pathway during T lymphocytes activation in mice was studied.The blank control group(excluding Con A),Con A(5μg·mL~(-1)) control group,ZEA(10,20 and 40μmol·L~(-1))+Con A(5μg·mL~(-1)) treatment group were established.The cells were observed and photographed by transmission electron microscopy 48 hours later.After 24 and 36hours of exposure,the cytoplasm and nuclear proteins were extracted respectively.Western blot was used to detect the expression of NFAT,NF-κB and other proteins in cytoplasm and nucleus.The results showed that the expression of NFAT,NF-κB and other proteins in the cytoplasm of Con A group cells were significantly higher than in the blank control group cells(excluding Con A)(P<0.05 or P<0.01).The volume of T lymphocytes increased,which contains a large number of cytoplasm,ribosomes and mitochondria.Compared with the blank group,the contents of NFAT,NF-κB in the nucleus of T lymphocytes of Con A group were significantly increased(P<0.01).With the increase of ZEA exposure concentration,the expression of NFAT,NF-κB and other proteins in the cytoplasm of ZEA group cells were significantly lower than in the blank control group cells(excluding Con A)(P<0.05 or P<0.01).The expression levels of NFAT,NF-κB in nucleus of Con A group cells were obviously higher than in the blank control group cells(excluding Con A)(P<0.01).ZEA can damage the ultrastructure of T lymphocytes during activation and inhibit the activity of NFAT,NF-κB pathway.ZEA can inhibit the synthesis of related proteins and the activation of T lymphocytes.
To reveal the mechanism of zearalenone(ZEA)on immune function in mice,the primary spleen lymphocytes stimulated by Concanavalin A(Con A)were used as the experiment material.The effect of ZEA on the ultrastructure and NFAT,NF-κB signaling pathway during T lymphocytes activation in mice was studied.The blank control group(excluding Con A),Con A(5μg·mL~(-1)) control group,ZEA(10,20 and 40μmol·L~(-1))+Con A(5μg·mL~(-1)) treatment group were established.The cells were observed and photographed by transmission electron microscopy 48 hours later.After 24 and 36hours of exposure,the cytoplasm and nuclear proteins were extracted respectively.Western blot was used to detect the expression of NFAT,NF-κB and other proteins in cytoplasm and nucleus.The results showed that the expression of NFAT,NF-κB and other proteins in the cytoplasm of Con A group cells were significantly higher than in the blank control group cells(excluding Con A)(P<0.05 or P<0.01).The volume of T lymphocytes increased,which contains a large number of cytoplasm,ribosomes and mitochondria.Compared with the blank group,the contents of NFAT,NF-κB in the nucleus of T lymphocytes of Con A group were significantly increased(P<0.01).With the increase of ZEA exposure concentration,the expression of NFAT,NF-κB and other proteins in the cytoplasm of ZEA group cells were significantly lower than in the blank control group cells(excluding Con A)(P<0.05 or P<0.01).The expression levels of NFAT,NF-κB in nucleus of Con A group cells were obviously higher than in the blank control group cells(excluding Con A)(P<0.01).ZEA can damage the ultrastructure of T lymphocytes during activation and inhibit the activity of NFAT,NF-κB pathway.ZEA can inhibit the synthesis of related proteins and the activation of T lymphocytes.
引文
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[24]JIMI E,GHOSH S.Role of nuclear factor-kappaB in the immune system and bone[J].Immunological Reviews,2005,208(3):80-87.
[2]DORIC M,RADOVIC S,BABIC M,et al.Zearalenone-induced lymphophagocytosis(T cell apoptosis)on the rat’s thymus[J].Bosnian Journal of Basic Medical Sciences,2007,7(1):66-70.
[3]VAN DE PAVERT S A,MEBIUS R E.New insights into the development of lymphoid tissues[J].Nature Reviews Immunology,2010,10(9):664-674.
[4]PURNER M B,BERENS R L,TOMAVO S,et al.Stimulation of human T lymphocytes obtained from Toxoplasma gondii-seronegative persons by proteins derived from T.gondii[J].Journal of Infectious Diseases,1998,177(3):746-753.
[5]LEE K H,HOLDORF A D,DUSTIN M L,et al.T cell receptor signaling precedes immunological synapse formation[J].Science,2002,295(5559):1539-1542.
[6]AIFANTIS I,GOUNARI F,SCORRANO L,et al.Constitutive pre-TCR signaling promotes differentiation through Ca2+mobilization and activation of NF-kappaB and NFAT[J].Nature Immunology,2001,2(5):403-409.
[7]WONG V K,ZHOU H,CHEUNG S S,et al.Mechanistic study of saikosaponin-d(Ssd)on suppression of murine T lymphocyte activation[J].Journal of Cellular Biochemistry,2009,107(2):303-315.
[8]GHOSH S,KARIN M.Missing Pieces in the NF-kappaB puzzle[J].Cell,2002,109(2):S81-S96.
[9]KARIN M,GRETEN F R.NF-kappaB:linking inflammation and immunity to cancer development and progression[J].Nature Reviews Immunology,2005,5(10):749-759.
[10]SHAW J P,UTZ P J,DURAND D B,et al.Identification of a putative regulator of early T cell activation genes[J].Science,1988,241(4862):202-205.
[11]DE GREGORIO R,I珦NIGUEZ M A,FRESNO M,et al.Cot kinase induces cyclooxygenase-2expression in T cells through activation of the nuclear factor of activated T cells[J].Journal of Biological Chemistry,2001,276(29):27003-27009.
[12]FRASER J H,RINCN M,MCCOY K D,et al.CTLA4ligation attenuates AP-1,NFAT and NF-kappaB activity in activated T cells[J].European Journal of Immunology,1999,29(3):838-844.
[13]KRAUSS S,BUTTGEREIT F,BRAND M D.Effects of the mitogen concanavalin A on pathways of thymocyte energy metabolism[J].Biochimica et Biophysica Acta(BBA)-Bioenergetics,1999,1412(2):129-138.
[14]蔡国栋,孙凯,项自来,等.玉米赤霉烯酮对小鼠T淋巴细胞体外活化、增殖的影响[J].畜牧兽医学报,2017,48(7):1357-1364.
[15]MINERVINI F,DELL’AQUILA M E.Zearalenone and reproductive function in farm animals[J].International Journal of Molecular Sciences,2008,9(12):2570-2584.
[16]ZHAO F,LI R,XIAO S,et al.Postweaning exposure to dietary zearalenone,a mycotoxin,promotes premature onset of puberty and disrupts early pregnancy events in female mice[J].Toxicological Sciences,2013,132(2):431-442.
[17]董双,何剑斌,张燚,等.玉米赤霉烯酮对雄性小鼠生殖系统的毒性作用[J].畜牧与兽医,2016,48(6):25-29.
[18]MARIN D E,TARANU I,BURLACU R,et al.Effects of zearalenone and its derivatives on porcine immune response[J].Toxicology in Vitro,2011,25(8):1981-1988.
[19]VLATA Z,PORICHIS F,TZANAKAKIS G,et al.A study of zearalenone cytotoxicity on human peripheral blood mononuclear cells[J].Toxicology Letters,2006,165(3):274-281.
[20]WU Y Q,BORDE M,HEISSMEYER V,et al.FOXP3controls regulatory T cell function through cooperation with NFAT[J].Cell,2006,126(2):375-387.
[21]CRABTREE G R,OLSON E N.NFAT signaling:choreographing the social lives of cells[J].Cell,2002,109(2):S67-S79.
[22]PENG S L,GERTH A J,RANGER A M,et al.NFATc1and NFATc2together control both T and B cell activation and differentiation[J].Immunity,2001,14(1):13-20.
[23]FESKE S,GILTNANE J,DOLMETSCH R,et al.Gene regulation mediated by calcium signals in T lymphocytes[J].Nature Immunology,2001,2(4):316-324.
[24]JIMI E,GHOSH S.Role of nuclear factor-kappaB in the immune system and bone[J].Immunological Reviews,2005,208(3):80-87.
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