自噬对DON诱导仔猪海马神经细胞损伤的影响
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摘要
[目的]本试验旨在研究自噬对脱氧雪腐镰刀菌烯醇(DON)诱导仔猪海马神经细胞损伤的影响。[方法]以不同质量浓度DON(0,125,250,500,1 000和2 000 ng·mL~(-1))处理对数生长期仔猪海马神经细胞24 h后,采用倒置显微镜、透射电镜观察DON对仔猪海马神经细胞形态和超微结构的影响;采用p EGFP-LC3质粒瞬时转染细胞,荧光显微镜检测绿色荧光蛋白的分布情况;采用Western blot检测DON对仔猪海马神经细胞自噬标志性蛋白LC3的表达;通过添加自噬激活剂雷帕霉素(RAP)和自噬抑制剂氯喹(CQ)来升高和降低细胞的自噬水平,CCK-8法检测细胞的存活率。[结果]不同质量浓度的DON均可诱导细胞自噬,自噬水平随着DON浓度的增加先上升后下降,在1 000 ng·mL~(-1)时达到峰值。与对照组相比,DON可显著抑制细胞存活率并诱导细胞发生明显的形态学变化,并呈剂量依赖性;随着DON浓度的升高,细胞LC3-Ⅱ含量先上升后下降,1 000 ng·mL~(-1)时,LC3-Ⅱ含量最高,差异显著或极显著(P<0.05或P<0.01)。与DON处理组相比,自噬水平的上升显著提高细胞活性(P<0.05),而自噬水平的下降显著降低细胞活性(P<0.05)。[结论]DON可以引起仔猪海马神经细胞自噬水平的上升,激活的自噬对DON导致的细胞损伤具有一定的保护作用。
[Objectives]The aim of the study was to investigate the effects of autophagy on piglet hippocampal nerve cells( PHNC)damaged by deoxynivalenol( DON). [Methods]Hippocampal nerve cells were exposed to DON at different mass concentrations( 0,125,250,500,1 000 and 2 000 ng·mL~(-1)) for 24 h. The variations in cell morphology and cell ultrastructure were observed by inverted microscopy and transmission electron microscopy respectively. Transiently transfected with p EGFP-LC3 plasmid in PHNC and photomicrographs of GFP-LC3 were obtained by fluorescence microscopy. Microtubule-associated protein 1 light chain 3( LC3) indicator of autophagy was examined by Western blot. After rapamycin( RAP) and chloroquine( CQ) induced an increased and decreased level of autophagy,the effects of different levels of autophagy on cell viability were examined by cell counting kit-8( CCK-8) method. [Results]Different mass concentrations of DON induced cell autophagy. Compared with control group,the cell autophagy appearance in treatment groups significantly increased and then decreased. It also showed changes with the increasing of DON concentrations and reached peak level at 1 000 ng·mL~(-1). Compared with the control group,DON treatment inhibited cell proliferation as well as induced significant morphological changes with dose dependent manner. During comparison with the control group,when the concentration of DON increased,the levels of LC3-Ⅱ proteins significantly increased and then decreased with the highest level of 1 000 ng·mL~(-1)( P<0.05 or P<0.01). Compared with the DON treatment group,the increased level of autophagy significantly improved the viability of cells( P<0.05) and the decreased level of autophagy reduced the viability of cells significantly( P<0.05). [Conclusions]DON can increase the level of autophagy in PHNC,and activated autophagy serves as an important protective role against DON-induced cytotoxicity.
[Objectives]The aim of the study was to investigate the effects of autophagy on piglet hippocampal nerve cells( PHNC)damaged by deoxynivalenol( DON). [Methods]Hippocampal nerve cells were exposed to DON at different mass concentrations( 0,125,250,500,1 000 and 2 000 ng·mL~(-1)) for 24 h. The variations in cell morphology and cell ultrastructure were observed by inverted microscopy and transmission electron microscopy respectively. Transiently transfected with p EGFP-LC3 plasmid in PHNC and photomicrographs of GFP-LC3 were obtained by fluorescence microscopy. Microtubule-associated protein 1 light chain 3( LC3) indicator of autophagy was examined by Western blot. After rapamycin( RAP) and chloroquine( CQ) induced an increased and decreased level of autophagy,the effects of different levels of autophagy on cell viability were examined by cell counting kit-8( CCK-8) method. [Results]Different mass concentrations of DON induced cell autophagy. Compared with control group,the cell autophagy appearance in treatment groups significantly increased and then decreased. It also showed changes with the increasing of DON concentrations and reached peak level at 1 000 ng·mL~(-1). Compared with the control group,DON treatment inhibited cell proliferation as well as induced significant morphological changes with dose dependent manner. During comparison with the control group,when the concentration of DON increased,the levels of LC3-Ⅱ proteins significantly increased and then decreased with the highest level of 1 000 ng·mL~(-1)( P<0.05 or P<0.01). Compared with the DON treatment group,the increased level of autophagy significantly improved the viability of cells( P<0.05) and the decreased level of autophagy reduced the viability of cells significantly( P<0.05). [Conclusions]DON can increase the level of autophagy in PHNC,and activated autophagy serves as an important protective role against DON-induced cytotoxicity.
引文
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[30] Yang L Y,Wu K H,Chiu W T,et al. The cadmium-induced death of mesangial cells results in nephrotoxicity[J]. Autophagy,2009,5(4):571-572.
[2] Kim Y C,Guan K L. mT OR:a pharmacologic target for autophagy regulation[J]. Journal of Clinical Investigation,2015,125(1):25-32.
[3] Clark S L,Jr. Cellular differentiation in the kidneys of newborn mice studied with the electron microscope[J]. The Journal of Biophysical and Biochemical Cytology,1957,3(3):349-362.
[4] Yoshimori T. Autophagy:a regulated bulk degradation process inside cells[J]. Biochemical&Biophysical Research Communications,2004,313(2):453-458.
[5] Nakatogawa H,Suzuki K,Kamada Y,et al. Dynamics and diversity in autophagy mechanisms:lessons from yeast[J]. Nature Reviews Molecular Cell Biology,2009,10(7):458-467.
[6] Nakagawa I,Amano A,Mizushima N,et al. Autophagy defends cells against invading group A Streptococcus[J]. Science,2004,306(5698):1037-1040.
[7] Orvedahl A,Levine B. Autophagy in mammalian antiviral immunity[M]//Levine B,Yoshimori T,Deretic V. Current Topics in Microbiology and Immunology. Berlin:Springer,2009:267-285.
[8] English L,Chemali M,Duron J,et al. Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1infection[J]. Nature Immunology,2009,10(7):480-487.
[9] Fimia G M,Stoykova A,Romagnoli A,et al. Ambra1 regulates autophagy and development of the nervous system[J]. Nature,2007,447(7148):1121-1125.
[10] Singh R,Kaushik S,Wang Y,et al. Autophagy regulates lipid metabolism[J]. Nature,2009,458(7242):1131-1135.
[11]谢俊泽,杨小康,严磊,等.自噬在镉致BRL3A细胞损伤中的保护作用[J].中国兽医学报,2016,36(11):1899-1902.Xie J Z,Yang X K,Yan L,et al. Induction of cytoprotective autophagy in BRL3A cells by cadmium[J]. Journal of Chinese Veterinary Science,2016,36(11):1899-1902(in Chinese with English abstract).
[12] Ng S,Wu Y T,Chen B,et al. Impaired autophagy due to constitutive mT OR activation sensitizes TSC2-null cells to cell death under stress[J].Autophagy,2011,7(10):1173-1186.
[13] Savard C,Nogues P,Boyer A,et al. Prevention of deoxynivalenol-and zearalenone-associated oxidative stress does not restore MA-10 Leydig cell functions[J]. Toxicology,2016,341/342/343:17-27.
[14] Liu Y P,Lu Y,Wang L Y,et al. Occurrence of deoxynivalenol in wheat,Hebei Province,China[J]. Food Chemistry,2016,197:1271-1274.
[15] Park S H,Kim J,Kim D,et al. Mycotoxin detoxifiers attenuate deoxynivalenol-induced pro-inflammatory barrier insult in porcine enterocytes as an in vitro evaluation model of feed mycotoxin reduction[J]. Toxicology in Vitro,2016,38:108-116.
[16]范梦雪,陈晓芳,姜云晶,等.脱氧雪腐镰刀菌烯醇对仔猪海马神经细胞的毒性作用[J].中国兽医科学,2017,47(1):121-127.Fan M X,Chen X F,Jiang Y J,et al. Toxic effects of deoxynivalenol on hippocampal nerve cells of piglets[J]. Chinese Veterinary Science,2017,47(1):121-127(in Chinese with English abstract).
[17]耿芳芳,许伟,范梦雪,等.脱氧雪腐镰刀菌烯醇对雏鸡脂质过氧化反应及脑部形态结构的影响[J].南京农业大学学报,2016,39(3):460-466. DOI:10.7685/jnau.201510024.Geng F F,Xu W,Fan M X,et al. Effects of deoxynivalenol on lipid peroxidation reaction and brain morphology in chicken[J]. Journal of Nanjing Agricuitural University,2016,39(3):460-466(in Chinese with English abstract).
[18]耿芳芳,范梦雪,姜云晶,等. DON暴露对雏鸡神经递质及钙调蛋白含量的影响[J].西北农林科技大学学报(自然科学版),2017,45(2):56-62.Geng F F,Fan M X,Jiang Y J,et al. Effects of deoxynivalenol exposure on neurotransmitter and calmodulin contents in chicken[J]. Journal of Northwest A&F University(Natural Science Edition),2017,45(2):56-62(in Chinese with English abstract).
[19] Bensassi F,Gallerne C,Dein O S E,et al. Involvement of mitochondria-mediated apoptosis in deoxynivalenol cytotoxicity[J]. Food and Chemical Toxicology,2012,50(5):1680-1689.
[20] Savard C,Pinilla V,Provost C,et al. In vitro,effect of deoxynivalenol(DON)mycotoxin on porcine reproductive and respiratory syndrome virus replication[J]. Food&Chemical Toxicology,2014,65(1):219-226.
[21]陈晓芳,张娅菲,储小燕,等.脱氧雪腐镰刀菌烯醇对仔猪海马神经细胞凋亡及线粒体膜电位的影响[J].南京农业大学学报,2018,41(3):526-533. DOI:10.7685/jnau.201705035.Chen X F,Zhang Y F,Chu X Y,et al. Effects of deoxynivalenol on apoptosis and mitochondrial membrane potential in piglet hippocampal nerve cells[J]. Journal of Nanjing Agricuitural University,2018,41(3):526-533(in Chinese with English abstract).
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[23] Poornima P,Weng C F,Padma V V. Neferine from Nelumbo nucifera induces autophagy through the inhibition of PI3K/Akt/mT OR pathway and ROS hyper generation in A549 cells[J]. Food Chemistry,2013,141(4):3598-3605.
[24] Codogno P,Meijer A J. Autophagy and signaling:their role in cell survival and cell death[J]. Cell Death Differ,2005,12(2):1509-1518.
[25] Han J,Wang Q C. Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation[J].Toxicology and Applied Pharmacology,2016,300:70-76.
[26] Edinger A L,Thompson C B. Death by design:apoptosis,necrosis and autophagy[J]. Current Opinion in Cell Biology,2004,16(6):663-669.
[27] Xia J,Guo S,Fang T,et al. Dihydromyricetin induces autophagy in HepG2 cells involved in inhibition of mT OR and regulating its upstream pathways[J]. Food&Chemical Toxicology,2014,66(4):7-13.
[28]司梦雪,王冰洁,冯楠楠,等.自噬在玉米赤霉烯酮诱导TM4细胞凋亡中的作用[J].畜牧与兽医,2018,50(8):104-109.Si M X,Wang B J,Feng N N,et al. Effect of autophagy on apoptosis induced by zearalenone in TM4 cells[J]. Animal Husbandry&Veterinary Medicine,2018,50(8):104-109(in Chinese with English abstract).
[29] Zhang H,Gong Y,Wang Z,et al. Apelin inhibits the proliferation and migration of rat PASMCs via the activation of PI3K/Akt/mT OR signal and the inhibition of autophagy under hypoxia[J]. Journal of Cellular&Molecular Medicine,2014,18(3):542-553.
[30] Yang L Y,Wu K H,Chiu W T,et al. The cadmium-induced death of mesangial cells results in nephrotoxicity[J]. Autophagy,2009,5(4):571-572.