玉米赤霉烯酮对鸡胚成纤维细胞的毒性作用

doi:10.11843/j.issn.0366-6964.2022.05.029

摘要/Abstract

摘要： 旨在探究玉米赤霉烯酮(ZEA)对鸡胚成纤维细胞(DF-1)的毒性作用机制，采用MTT法检测细胞活力变化，比色法检测乳酸脱氢酶(LDH)活力，ELISA法检测Caspase-3含量，Annexin V-FITC/PI双染法检测细胞凋亡，荧光显微镜观察细胞活性氧(ROS)水平，线粒体膜电位变化，RT-qPCR法检测内质网应激(ERs)和细胞凋亡相关基因的mRNA转录水平。结果显示，12.5~50.0 μg·mL^-1 ZEA显著抑制DF-1细胞增殖(P<0.01)，且呈时间和剂量依赖性关系。25.0 μg·mL^-1 ZEA处理细胞24 h后，上清液中LDH和Caspase-3含量显著升高(P<0.01)；细胞中ROS水平和细胞凋亡数量极显著升高(P<0.01)；线粒体膜电位极显著降低(P<0.01)。凋亡相关基因Caspase-3、Bax mRNA转录水平极显著上调(P<0.01)，Bcl-2 mRNA转录水平极显著下调(P<0.01)；ERs相关基因GRP78、ATF6、ATF4、CHOP、PERK mRNA转录水平极显著上调(P<0.01)。综上表明，ZEA能通过内质网应激途径导致细胞凋亡并对鸡胚成纤维细胞发挥毒性作用。研究结果为深入研究ZEA对鸡细胞的毒性作用和解毒手段奠定基础，对相关禽类疾病治疗具有意义。

关键词: 玉米赤霉烯酮, 鸡胚成纤维细胞, 细胞毒性, 内质网应激, 细胞凋亡

Abstract: This study was conducted to investigate the mechanism of the toxic effects of zearalenone (ZEA) on chicken embryo fibroblasts (DF-1). The cell viability, lactate dehydrogenase activity, Caspase-3 levels, cell apoptosis, cellular reactive oxygen species levels, mitochondrial membrane potential change and expression of endoplasmic reticulum stress (ERs) and apoptosis-related genes were detected by MTT, colorimetric assay, ELISA, Annexin V-FITC/PI staining, fluorescence microscopy and RT-qPCR, respectively. The results showed that 12.5-50.0 μg·mL^-1 ZEA significantly inhibited the proliferation of DF-1 cells (P<0.01) in a time- and dose-dependent manner. Herein 25.0 μg·mL^-1 ZEA treated cells for 24 h resulted in a significant increase in LDH and Caspase-3 levels in the supernatant (P<0.01); the levels of ROS and the number of apoptotic cells in the cells were highly significant (P<0.01); the mitochondrial membrane potential was significantly reduced (P<0.01). The mRNA expression levels of apoptosis-related genes Caspase-3 and Bax were highly significantly up-regulated (P<0.01) and that of Bcl-2 was highly significantly down-regulated (P<0.01); the mRNA expression levels of ERs-related genes GRP78, ATF6, ATF4, CHOP and PERK were highly significantly up-regulated (P<0.01). The results indicated that ZEA could exert toxic effects on chicken embryonic fibroblasts through endoplasmic reticulum stress leading to apoptosis. The results of the study provide a basis for further research on the toxic effects of ZEA on chicken cells and the means of detoxification, and have implications for the treatment of related avian diseases.

Key words: zearalenone, chicken embryo fibroblasts, cytotoxicity, endoplasmic reticulum stress, apoptosis

中图分类号:

S859.82

张凯照, 胡会, 许泽锴, 王诗倩, 崔红杰, 黄小红. 玉米赤霉烯酮对鸡胚成纤维细胞的毒性作用[J]. 畜牧兽医学报, 2022, 53(5): 1615-1625.

ZHANG Kaizhao, HU Hui, XU Zekai, WANG Shiqian, CUI Hongjie, HUANG Xiaohong. Toxicity of Zearalenone on Chicken Embryo Fibroblasts[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1615-1625.

参考文献 35

[1]	闫昭明, 陈清华, 陈凤鸣. 玉米赤霉烯酮毒性研究[J]. 动物营养学报, 2018, 30(9):3453-3458.YAN Z M, CHEN Q H, CHEN F M. Study on toxicity of zearalenone[J]. Chinese Journal of Animal Nutrition, 2018, 30(9):3453-3458. (in Chinese)
[2]	LEE H J, RYU D. Worldwide occurrence of mycotoxins in cereals and cereal-derived food products:public health perspectives of their co-occurrence[J]. J Agric Food Chem, 2017, 65(33):7034-7051.
[3]	黄俊恒, 黄广明. 2018年19省市饲料及饲料原料霉菌毒素污染状况分析[J]. 养猪, 2019(3):6-8.HUANG J H, HUANG G M. Analysis on mycotoxin contamination of feed and feed materials in 19 provinces and cities in 2018[J]. Swine Prod, 2019(3):6-8. (in Chinese)
[4]	BELHASSEN H, JIMÉNEZ-DÍAZ I, GHALI R, et al. Validation of a UHPLC-MS/MS method for quantification of zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol and zearalanone in human urine[J]. J Chromatogr B, 2014, 962:68-74.
[5]	ZHENG W L, FENG N N, WANG Y, et al. Effects of zearalenone and its derivatives on the synthesis and secretion of mammalian sex steroid hormones:a review[J]. Food Chem Toxicol, 2019, 126:262-276.
[6]	HAQUE A, WANG Y H, SHEN Z Q, et al. Mycotoxin contamination and control strategy in human, domestic animal and poultry:a review[J]. Microb Pathog, 2020, 142:104095.
[7]	WANG Y L, ZHOU X Q, JIANG W D, et al. Effects of dietary zearalenone on oxidative stress, cell apoptosis, and tight junction in the intestine of juvenile grass carp (Ctenopharyngodon idella)[J]. Toxins, 2019, 11(6):333.
[8]	梁梓森, 马勇江, 刘长永, 等. 玉米赤霉烯酮对小鼠肝脏及肾脏的毒性作用[J]. 中国兽医学报, 2010, 30(5):673-676.LIANG Z S, MA Y J, LIU C Y, et al. In vivo toxicity of zearalenone on liver and kidney in mice[J]. Chinese Journal of Veterinary Science, 2010, 30(5):673-676. (in Chinese)
[9]	BULGARU C V, MARIN D E, PISTOL G C, et al. Zearalenone and the immune response[J]. Toxins, 2021, 13(4):248.
[10]	李欣虹, 郑若愚, 辜彦霏, 等. 玉米赤霉烯酮对体外培养鸡脾脏淋巴细胞凋亡的影响[J]. 动物营养学报, 2018, 30(8):3285-3292.LI X H, ZHENG R Y, GU Y F, et al. Effects of zearalenone on apoptosis of chicken splenic lymphocytes in vitro[J]. Chinese Journal of Animal Nutrition, 2018, 30(8):3285-3292. (in Chinese)
[11]	CAI G D, SI M X, LI X, et al. Zearalenone induces apoptosis of rat Sertoli cells through Fas-Fas ligand and mitochondrial pathway[J]. Environ Toxicol, 2019, 34(4):424-433.
[12]	裴亚萍, 赵瑾, 孙娜, 等. 咖啡酸对玉米赤霉烯酮诱导小鼠卵巢颗粒细胞凋亡的保护作用[J]. 畜牧兽医学报, 2020, 51(12):3068-3075.PEI Y P, ZHAO J, SUN N, et al. The effect of caffeic acid on zearalenone-induced ovarian granulosa cell apoptosis in mice[J]. Acta Veterinaria et Zootechnica Scinia, 2020, 51(12):3068-3075. (in Chinese)
[13]	WANG X, YU H, FANG H T, et al. Transcriptional profiling of zearalenone-induced inhibition of IPEC-J2 cell proliferation[J]. Toxicon, 2019, 172:8-14.
[14]	WANG Y J, ZHENG W L, BIAN X J, et al. Zearalenone induces apoptosis and cytoprotective autophagy in primary Leydig cells[J]. Toxicol Lett, 2014, 226(2):182-191.
[15]	杨桂然, 王福科, 李彦林. 成纤维细胞的生物学特性及分化潜能[J]. 中国组织工程研究, 2020, 24(13):2114-2119.YANG G R, WANG F K, LI Y L. Biological characteristics and differentiation potential of fibroblasts[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(13):2114-2119. (in Chinese)
[16]	许巧. 镰刀菌毒素对动物毒性作用的研究进展[J]. 中国畜牧杂志, 2021, 57(4):61-67.XU Q. Research progress on toxicity of fusarium mycotoxins in animals[J]. Chinese Journal of Animal Science, 2021, 57(4):61-67. (in Chinese)
[17]	LYNCH M D, WATT F M. Fibroblast heterogeneity:implications for human disease[J]. J Clin Invest, 2018, 128(1):26-35.
[18]	FANTIN V R, ST-PIERRE J, LEDER P. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance[J]. Cancer Cell, 2006, 9(6):425-434.
[19]	陈新亮. 原花青素对玉米赤霉烯酮致MODE-K细胞氧化损伤及内质网应激凋亡保护作用研究[D]. 沈阳:沈阳农业大学, 2018.CHEN X L. Protective effect of proanthocyanidins on the oxidative damage and apoptosis of endoplasmic reticulum stress of MODE-K cells induced by zearalenone[D]. Shenyang:Shenyang Agricultural University, 2018. (in Chinese)
[20]	ZOROV D B, JUHASZOVA M, SOLLOTT S J. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release[J]. Physiol Rev, 2014, 94(3):909-950.
[21]	HASSEN W, AYED-BOUSSEMA I, OSCOZ A A, et al. The role of oxidative stress in zearalenone-mediated toxicity in Hep G2 cells:oxidative DNA damage, gluthatione depletion and stress proteins induction[J]. Toxicology, 2007, 232(3):294-302.
[22]	SINHA K, DAS J, PAL P B, et al. Oxidative stress:the mitochondria-dependent and mitochondria-independent pathways of apoptosis[J]. Arch Toxicol, 2013, 87(7):1157-1180.
[23]	张心怡, 吴云平, 蔡国栋, 等. 玉米赤霉烯酮对小鼠T淋巴细胞活性氧及线粒体膜电位的影响[J]. 畜牧与兽医, 2018, 50(8):90-94.ZHANG X Y, WU Y P, CAI G D, et al. Effects of ZEA on reactive oxygen species and mitochondrial membrane potential in T lymphocytes of mice[J]. Animal Husbandry & Veterinary Medicine, 2018, 50(8):90-94. (in Chinese)
[24]	RON D, WALTER P. Signal integration in the endoplasmic reticulum unfolded protein response[J]. Nat Rev Mol Cell Biol, 2007, 8(7):519-529.
[25]	COLEMAN O I, HALLER D. ER stress and the UPR in shaping intestinal tissue homeostasis and immunity[J]. Front Immunol, 2019, 10:2825.
[26]	王宗捷, 张瑞雪, 刘守勤, 等. 玉米赤霉烯酮诱导山羊子宫内膜基质细胞凋亡的研究[J]. 畜牧兽医学报, 2021, 52(2):535-542.WANG Z J, ZHANG R X, LIU S Q, et al. The apoptotic effect of zearalenone on goat endometrial stromal cells[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(2):535-542. (in Chinese)
[27]	PARK S W, OZCAN U. Potential for therapeutic manipulation of the UPR in disease[J]. Semin Immunopathol, 2013, 35(3):351-373.
[28]	BAUMEISTER P, LUO S Z, SKARNES W C, et al. Endoplasmic reticulum stress induction of the Grp78/BiP promoter:activating mechanisms mediated by YY1 and its interactive chromatin modifiers[J]. Mol Cell Biol, 2005, 25(11):4529-4540.
[29]	杨方晓, 李莲, 赵若含, 等. 褪黑素对HT-2毒素诱导的牛卵巢颗粒细胞内质网应激与自噬的影响[J]. 南京农业大学学报, 2020, 43(1):143-150.YANG F X, LI L, ZHAO R H, et al. Effects of melatonin on endoplasmic reticulum stress and autophagy in bovine ovarian granulosa cells induced by HT-2 toxin[J]. Journal of Nanjing Agricultural University, 2020, 43(1):143-150. (in Chinese)
[30]	SU C M, WANG S W, LEE T H, et al. Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells[J]. Toxicol Appl Pharmacol, 2013, 272(2):335-344.
[31]	GAN Z, JIANG K F, WU H C, et al. Polydatin reduces Staphylococcus aureus lipoteichoic acid-induced injury by attenuating reactive oxygen species generation and TLR2-NFκB signalling[J]. J Cell Mol Med, 2017, 21(11):2796-2808.
[32]	PEÑA-BLANCO A, GARCÍA-SÁEZ A G. Bax, Bak and beyond-mitochondrial performance in apoptosis[J]. FEBS J, 2018, 285(3):416-431.
[33]	NAIM S, KAUFMANN T. The multifaceted roles of the BCL-2 family member BOK[J]. Front Cell Dev Biol, 2020, 8:574338.
[34]	VARDIYAN R, EZATI D, ANVARI M, et al. Effect of L-carnitine on the expression of the apoptotic genes Bcl-2 and Bax[J]. Clin Exp Reprod Med, 2020, 47(3):155-160.
[35]	SURESH K, CARINO K, JOHNSTON L, et al. A nonapoptotic endothelial barrier-protective role for caspase-3[J]. Am J Physiol-Lung Cell Mol Physiol, 2019, 316(6):L1118-L1126.