玉米赤霉烯酮对不同时期小鼠子宫细胞凋亡及凋亡因子的影响
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摘要
本试验旨在研究在饲粮中添加不同水平的玉米赤霉烯酮(ZEA)对发情前期、发情期、产仔后雌性小鼠子宫细胞凋亡及相关因子的影响。选取4周龄昆明雌性小鼠120只,随机分为4组,每组30只。选取雄性小鼠20只,随机分为4组,每组5只。7 d预试期结束后,试验分2个阶段。第1阶段,各组雌鼠分别饲喂在基础饲粮中添加0(对照)、50、100、150 mg/kg ZEA的饲粮,饲喂时间是20 d。饲养试验结束后的第2、3、4天,通过阴道涂片法每组挑选出发情前期、发情期小鼠各10只,脱椎处死,摘取子宫角,用于子宫细胞凋亡原位末端标记法(TUNEL)染色和凋亡相关因子mRNA表达量的检测。第2阶段,每组剩余雌鼠与雄鼠按2∶1进行合笼,至产仔后,按上述方法采样检测。结果表明:1)发情前期凋亡细胞主要位于子宫内膜间质细胞及上皮细胞;与对照组相比,添加100、150 mg/kg ZEA极显著提高凋亡阳性表达细胞平均光密度值(P<0.01),且极显著上调半胱天冬蛋白酶(caspase)-3、caspase-8、caspase-9及B细胞淋巴瘤/白血病-2(Bcl-2) mRNA相对表达量(P<0.01); 2)发情期细胞凋亡主要集中在子宫内膜间质细胞、子宫内膜上皮细胞及子宫腺上皮细胞;与对照组相比,随着ZEA添加水平的增加,凋亡细胞呈剂量依赖性增加(P<0.01),能够极显著上调凋亡相关基因mRNA相对表达量(P<0.01); 3)产仔后期凋亡细胞分布较为均匀;与对照组相比,添加150 mg/kg ZEA凋亡程度极显著增加(P<0.01);添加100 mg/kg ZEA能够显著上调Bcl-2、caspase-3 mRNA相对表达量(P<0.05),添加150 mg/kg ZEA能够显著上调caspase-8、caspase-9 mRNA相对表达量(P<0.05)。综上所述,ZEA能够促进不同时期小鼠子宫细胞凋亡,并且能够上调凋亡相关基因caspase-3、caspase-8、caspase-9、Bcl-2表达。
The purpose of this study was to investigate the effects of different levels of zearalenone( ZEA) on the apoptosis of uterine cells and related factors in female mice in pre-estrus,estrus and post-partition periods.One hundred and twenty,4-week-old,female Kunming mice were randomly divided into 4 groups,30 in each group. Twenty male mice were randomly divided into 4 groups,5 in each group. After 7 days of pre-trial period,the experiment was divided into two stages. Stage 1: the mice of the four groups were fed the basal diets with 0,50,100 and 150 mg/kg ZEA for 20 days. At days 2,3 and 4 after the feeding test,ten mice in preestrus and estrus periods in each group were selected with vaginal smear. The selected mice were sacrificed by cervical delocation,and the uterine horns were harvested for TUNEL staining of apoptotic uterine cells and detection of apoptotic related factors mRNA expression. The remaining females of each group were caged with male mice by 2∶1. After giving birth,the samples were collected according to the above method. The results showed as follows: 1) apoptotic cells in pre-estrus period were mainly located in the endometrial stroma and epithelial cells. Compared with the control group,adding 100,150 mg/kg ZEA significantly increased the optical density of apoptotic cells( P<0.01),and significantly increased mRNA relative expression levels of the caspase-3,caspase-8,caspase-9 and B-cell lymphoma/leukemia-2( Bcl-2)( P<0.01). 2) In estrous period,apoptosis was observed mainly in endometrial stromal cells,endometrial epithelial cells and uterine gland cells;compared with the control group,with the increasing of ZEA level,apoptotic cells significantly increased( P<0.01),and the mRNA relative expression levels of apoptotic genes significantly increased( P<0.01). 3) The distribution of apoptotic cells were more uniform in the post-partition period; compared with the control group,adding 150 mg/kg ZEA significantly increased apoptosis( P<0.01). Meanwhile,adding 100 mg/kg ZEA significantly up-regulated the mRNA relative expression levels of Bcl-2 and caspase-3( P < 0.05),and adding150 mg/kg ZEA significantly up-regulated the mRNA relative expression levels of caspase-8 and caspase-9( P<0.05). In conclusion,ZEA promots the apoptosis of mice uterus in different periods and up-regulats the expression of apoptosis-related genes caspase-3,caspase-8,caspase-9 and Bcl-2.[Chinese Journal of Animal Nutrition,2019,31( 5) : 2388-2396]
The purpose of this study was to investigate the effects of different levels of zearalenone( ZEA) on the apoptosis of uterine cells and related factors in female mice in pre-estrus,estrus and post-partition periods.One hundred and twenty,4-week-old,female Kunming mice were randomly divided into 4 groups,30 in each group. Twenty male mice were randomly divided into 4 groups,5 in each group. After 7 days of pre-trial period,the experiment was divided into two stages. Stage 1: the mice of the four groups were fed the basal diets with 0,50,100 and 150 mg/kg ZEA for 20 days. At days 2,3 and 4 after the feeding test,ten mice in preestrus and estrus periods in each group were selected with vaginal smear. The selected mice were sacrificed by cervical delocation,and the uterine horns were harvested for TUNEL staining of apoptotic uterine cells and detection of apoptotic related factors mRNA expression. The remaining females of each group were caged with male mice by 2∶1. After giving birth,the samples were collected according to the above method. The results showed as follows: 1) apoptotic cells in pre-estrus period were mainly located in the endometrial stroma and epithelial cells. Compared with the control group,adding 100,150 mg/kg ZEA significantly increased the optical density of apoptotic cells( P<0.01),and significantly increased mRNA relative expression levels of the caspase-3,caspase-8,caspase-9 and B-cell lymphoma/leukemia-2( Bcl-2)( P<0.01). 2) In estrous period,apoptosis was observed mainly in endometrial stromal cells,endometrial epithelial cells and uterine gland cells;compared with the control group,with the increasing of ZEA level,apoptotic cells significantly increased( P<0.01),and the mRNA relative expression levels of apoptotic genes significantly increased( P<0.01). 3) The distribution of apoptotic cells were more uniform in the post-partition period; compared with the control group,adding 150 mg/kg ZEA significantly increased apoptosis( P<0.01). Meanwhile,adding 100 mg/kg ZEA significantly up-regulated the mRNA relative expression levels of Bcl-2 and caspase-3( P < 0.05),and adding150 mg/kg ZEA significantly up-regulated the mRNA relative expression levels of caspase-8 and caspase-9( P<0.05). In conclusion,ZEA promots the apoptosis of mice uterus in different periods and up-regulats the expression of apoptosis-related genes caspase-3,caspase-8,caspase-9 and Bcl-2.[Chinese Journal of Animal Nutrition,2019,31( 5) : 2388-2396]
引文
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[2] ZHANG K Z,TAN X T,LI Y,et al. Transcriptional profiling analysis of zearalenone-induced inhibition proliferation on mouse thymic epithelial cell line 1[J]. Ecotoxicology and Environmental Safety,2018,153:135-141.
[3] REDDY K E,JEONG J Y,LEE Y,et al. Deoxynivalenol-and zearalenone-contaminated feeds alter gene expression profiles in the livers of piglets[J]. AsianAustralasian Journal of Animal Sciences,2018,31(4):595-606.
[4] REDDY K E,LEE W,JEONG J Y,et al. Effects of deoxynivalenol-and zearalenone-contaminated feed on the gene expression profiles in the kidneys of piglets[J]. Asian-Australasian Journal of Animal Sciences,2018,31(1):138-148.
[5] MUTHULAKSHMI S,MAHARAJAN K,HABIBI H R,et al.Zearalenone induced embryo and neurotoxicity in zebrafish model(Danio rerio):role of oxidative stress revealed by a multi biomarker study[J].Chemosphere,2018,198:111-121.
[6] REN Z H,DENG H D,DENG Y T,et al.Combined effects of deoxynivalenol and zearalenone on oxidative injury and apoptosis in porcine splenic lymphocytes in vitro[J]. Experimental and Toxicologic Pathology,2017,69(8):612-617.
[7] CHEN X X,YANG C W,HUANG L B,et al.Zearalenone altered the serum hormones,morphologic and apoptotic measurements of genital organs in postw eaning gilts[J]. Asian-Australasian Journal of Animal Sciences,2015,28(2):171-179.
[8] MINERVINI F,DELL’AQUILA M E. Zearalenone and reproductive function in farm animals[J].International Journal of M olecular Sciences,2008,9(12):2570-2584.
[9]高爽,邓又天,张超,等.脱氧雪腐镰刀菌烯醇、玉米赤霉烯酮及其联合染毒对小鼠肝脏功能及肝细胞凋亡的影响[J].中国兽医学报,2015,35(12):2021-2026.
[10]张薇,张诗画,李建平,等.三种镰刀菌毒素对猪肾细胞凋亡及N-乙酰半胱胺酸保护作用的研究[C]//第二届(2017)东北养猪论坛暨东北猪业博览会论文集.哈尔滨:北京天可瀚传媒科技有限公司,2017:6.
[11]马勇江,许利娜,李玉谷,等.玉米赤霉烯酮对小鼠脾淋巴细胞凋亡的影响[J].家畜生态学报,2009,30(1):52-56.
[12]周迎芳.F-2毒素对小鼠卵巢颗粒细胞凋亡及其相关因素的影响研究[D].硕士学位论文.长沙:湖南农业大学,2007.
[13]胡进.玉米赤霉烯酮对小鼠子宫内膜基质细胞的毒性作用及机制研究[D].博士学位论文.南京:南京农业大学,2016.
[14] BEN SALEM I,PROLA A,BOUSSABBEH M,et al.Crocin and quercetin protect HCT116 and HEK293cells from zearalenone-induced apoptosis by reducing endoplasmic reticulum stress[J]. Cell Stress and Chaperones,2015,20(6):927-938.
[15] ZHENG W L,WANG B J,LI X,et al. Zearalenone promotes cell proliferation or causes cell death?[J].Toxins,2018,10(5):184.
[16] STOPA E,BABI N'SKA I,ZIELONKAL,et al. Immunohistochemical evaluation of apoptosis and proliferation in the mucous membrane of selected uterine regions in pre-pubertal bitches exposed to low doses of zearalenone[J].Polish Journal of Veterinary Sciences,2016,19(1):175-186.
[17] GAJECKA M,OBREMSKI K,JAKIMIUK E,et al.Histopathological examination of ovaries in bitches after experimental zearalenone mycotoxicosis[J]. Polish Journal of Veterinary Sciences,2008,11(4):363-366.
[18] YUAN H,DENG Y T,YUAN L Y,et al.Gynostemma pentaphyllum protects mouse male germ cells against apoptosis caused by zearalenone via Bax and Bcl-2regulation[J]. Toxicology M echanisms and M ethods,2010,20(3):153-158.
[19] ZHANG G L,SUN X F,FENG Y Z,et al.Zearalenone exposure impairs ovarian primordial follicle formation via dow n-regulation of Lhx8 expression in vitro[J]. Toxicology and Applied Pharmacology,2017,317:33-40.
[20] WANG J J,LI M M,ZHANG W,et al. Protective effect of N-acetylcysteine against oxidative stress induced by zearalenone via mitochondrial apoptosis pathw ay in SIEC02 cells[J]. Toxins,2018,10(10):407.
[21] SAMIK A,SAFITRI E.Potency of mycotoxin binders on M DA level,expressions of caspase 9 and caspase 3in the uterus of mice exposed to zearalenone[J]. Iraqi Journal of Veterinary Sciences,2017,31(1):29-33.
[22] SHI D H,ZHOU J C,ZHAO L H,et al.Alleviation of mycotoxin biodegradation agent on zearalenone and deoxynivalenol toxicosis in immature gilts[J]. Journal of Animal Science and Biotechnology,2018,9:42.
[23] LARANJEIRO C S M,DA SILVA L J G,PEREIRA A M P T,et al. The mycoestrogen zearalenone in portuguese flow ing w aters and its potential environmental impact[J].M ycotoxin Research,2018,34(1):77-83.
[24] LAHOUAR A,JEDIDI I,SANCHIS V,et al.Aflatoxin B1,ochratoxin A and zearalenone in sorghum grains marketed in tunisia[J]. Food Additives&Contaminants:Part B,2018,11(2):103-110.
[25] ZHOU M,YANG L J,SHAO M H,et al. Effects of zearalenone exposure on the TGF-β1/Smad3 signaling pathw ay and the expression of proliferation or apoptosis related genes of post-w eaning gilts[J]. Toxins(Basel),2018,10(2):E49.
[26] FLECK S C,CHURCHWELL M I,DOERGE D R.M etabolism and pharmacokinetics of zearalenone follow ing oral and intravenous administration in juvenile female pigs[J]. Food and Chemical Toxicology,2017,106:193-201.
[2] ZHANG K Z,TAN X T,LI Y,et al. Transcriptional profiling analysis of zearalenone-induced inhibition proliferation on mouse thymic epithelial cell line 1[J]. Ecotoxicology and Environmental Safety,2018,153:135-141.
[3] REDDY K E,JEONG J Y,LEE Y,et al. Deoxynivalenol-and zearalenone-contaminated feeds alter gene expression profiles in the livers of piglets[J]. AsianAustralasian Journal of Animal Sciences,2018,31(4):595-606.
[4] REDDY K E,LEE W,JEONG J Y,et al. Effects of deoxynivalenol-and zearalenone-contaminated feed on the gene expression profiles in the kidneys of piglets[J]. Asian-Australasian Journal of Animal Sciences,2018,31(1):138-148.
[5] MUTHULAKSHMI S,MAHARAJAN K,HABIBI H R,et al.Zearalenone induced embryo and neurotoxicity in zebrafish model(Danio rerio):role of oxidative stress revealed by a multi biomarker study[J].Chemosphere,2018,198:111-121.
[6] REN Z H,DENG H D,DENG Y T,et al.Combined effects of deoxynivalenol and zearalenone on oxidative injury and apoptosis in porcine splenic lymphocytes in vitro[J]. Experimental and Toxicologic Pathology,2017,69(8):612-617.
[7] CHEN X X,YANG C W,HUANG L B,et al.Zearalenone altered the serum hormones,morphologic and apoptotic measurements of genital organs in postw eaning gilts[J]. Asian-Australasian Journal of Animal Sciences,2015,28(2):171-179.
[8] MINERVINI F,DELL’AQUILA M E. Zearalenone and reproductive function in farm animals[J].International Journal of M olecular Sciences,2008,9(12):2570-2584.
[9]高爽,邓又天,张超,等.脱氧雪腐镰刀菌烯醇、玉米赤霉烯酮及其联合染毒对小鼠肝脏功能及肝细胞凋亡的影响[J].中国兽医学报,2015,35(12):2021-2026.
[10]张薇,张诗画,李建平,等.三种镰刀菌毒素对猪肾细胞凋亡及N-乙酰半胱胺酸保护作用的研究[C]//第二届(2017)东北养猪论坛暨东北猪业博览会论文集.哈尔滨:北京天可瀚传媒科技有限公司,2017:6.
[11]马勇江,许利娜,李玉谷,等.玉米赤霉烯酮对小鼠脾淋巴细胞凋亡的影响[J].家畜生态学报,2009,30(1):52-56.
[12]周迎芳.F-2毒素对小鼠卵巢颗粒细胞凋亡及其相关因素的影响研究[D].硕士学位论文.长沙:湖南农业大学,2007.
[13]胡进.玉米赤霉烯酮对小鼠子宫内膜基质细胞的毒性作用及机制研究[D].博士学位论文.南京:南京农业大学,2016.
[14] BEN SALEM I,PROLA A,BOUSSABBEH M,et al.Crocin and quercetin protect HCT116 and HEK293cells from zearalenone-induced apoptosis by reducing endoplasmic reticulum stress[J]. Cell Stress and Chaperones,2015,20(6):927-938.
[15] ZHENG W L,WANG B J,LI X,et al. Zearalenone promotes cell proliferation or causes cell death?[J].Toxins,2018,10(5):184.
[16] STOPA E,BABI N'SKA I,ZIELONKAL,et al. Immunohistochemical evaluation of apoptosis and proliferation in the mucous membrane of selected uterine regions in pre-pubertal bitches exposed to low doses of zearalenone[J].Polish Journal of Veterinary Sciences,2016,19(1):175-186.
[17] GAJECKA M,OBREMSKI K,JAKIMIUK E,et al.Histopathological examination of ovaries in bitches after experimental zearalenone mycotoxicosis[J]. Polish Journal of Veterinary Sciences,2008,11(4):363-366.
[18] YUAN H,DENG Y T,YUAN L Y,et al.Gynostemma pentaphyllum protects mouse male germ cells against apoptosis caused by zearalenone via Bax and Bcl-2regulation[J]. Toxicology M echanisms and M ethods,2010,20(3):153-158.
[19] ZHANG G L,SUN X F,FENG Y Z,et al.Zearalenone exposure impairs ovarian primordial follicle formation via dow n-regulation of Lhx8 expression in vitro[J]. Toxicology and Applied Pharmacology,2017,317:33-40.
[20] WANG J J,LI M M,ZHANG W,et al. Protective effect of N-acetylcysteine against oxidative stress induced by zearalenone via mitochondrial apoptosis pathw ay in SIEC02 cells[J]. Toxins,2018,10(10):407.
[21] SAMIK A,SAFITRI E.Potency of mycotoxin binders on M DA level,expressions of caspase 9 and caspase 3in the uterus of mice exposed to zearalenone[J]. Iraqi Journal of Veterinary Sciences,2017,31(1):29-33.
[22] SHI D H,ZHOU J C,ZHAO L H,et al.Alleviation of mycotoxin biodegradation agent on zearalenone and deoxynivalenol toxicosis in immature gilts[J]. Journal of Animal Science and Biotechnology,2018,9:42.
[23] LARANJEIRO C S M,DA SILVA L J G,PEREIRA A M P T,et al. The mycoestrogen zearalenone in portuguese flow ing w aters and its potential environmental impact[J].M ycotoxin Research,2018,34(1):77-83.
[24] LAHOUAR A,JEDIDI I,SANCHIS V,et al.Aflatoxin B1,ochratoxin A and zearalenone in sorghum grains marketed in tunisia[J]. Food Additives&Contaminants:Part B,2018,11(2):103-110.
[25] ZHOU M,YANG L J,SHAO M H,et al. Effects of zearalenone exposure on the TGF-β1/Smad3 signaling pathw ay and the expression of proliferation or apoptosis related genes of post-w eaning gilts[J]. Toxins(Basel),2018,10(2):E49.
[26] FLECK S C,CHURCHWELL M I,DOERGE D R.M etabolism and pharmacokinetics of zearalenone follow ing oral and intravenous administration in juvenile female pigs[J]. Food and Chemical Toxicology,2017,106:193-201.
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