亚硝酸盐胁迫对草鱼肝脏泛素-蛋白酶体系统的影响
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摘要
为了探究亚硝酸盐胁迫对草鱼(Ctenopharyngodon idellus)肝脏泛素-蛋白酶体系统(ubiquitin proteasome system,UPS)的影响,将草鱼随机暴露于0、0.5、20 mg/L的亚硝酸盐溶液中,在12、24、48、96 h后采集肝脏样品,检测血液的生化指标,nrf2、hsp70和UPS相关基因的表达,泛素化蛋白的含量以及肝脏的组织学变化。结果显示:在亚硝酸盐胁迫12 h,血清皮质醇含量呈现显著的剂量依赖式升高,且在24 h和48 h显著高于对照组。谷丙转氨酶和谷草转氨酶的含量在96 h显著性降低。暴露12 h时,20 mg/L暴露组的nrf2、hsp70的表达量显著性高于对照组,ub、chip、psma2、psmc的表达水平和泛素化蛋白的含量在20 mg/L亚硝酸盐胁迫24 h均出现显著升高。在高浓度亚硝酸盐胁迫96 h后,肝细胞出现明显的空泡化。研究表明,亚硝酸盐胁迫使草鱼产生了显著的应激反应,造成了肝组织损伤,使肝脏UPS活性上升。
Ctenopharyngodon idellus were randomly exposed to three nitrite concentrations(0,0.5,and 20.0 mg/L)for 96 h to evaluate the effect of nitrite stress on the ubiquitin-proteasome system(UPS)in the liver.The expression of genes,the content of ubiquitinated protein,histological alterations of liver,and the levels of serum biochemical parameters were investigated at 12,24,48 and 96 h after exposure,respectively.Compared to the control group,serum cortisol level was significantly elevated at 12 h after exposure in a dose-dependent manner.Meanwhile,the significant rising in serum cortisol level was detected at 24 and 48 h.Serum levels of alanine aminotransferase and aspartate aminotransferase were significantly decreased at 96 h.The mRNA levels of nrf2 and hsp70 increased significantly at 12 h after exposure(20.0 mg/L).Nitrite exposure of 20.0 mg/L caused the significant elevations in mRNA expressions of ub,chip,psmc1,and psma2 as well as in ubiquitinated protein content at 24 h.Hepatic vacuolization was observed in the liver of grass carp at 96 h after exposure to the concentration of nitrite(20.0 mg/L).These results suggested that the exposure of nitrite enhanced the activity of UPS as well as caused stress and histological damage of liver in C.idellus.
Ctenopharyngodon idellus were randomly exposed to three nitrite concentrations(0,0.5,and 20.0 mg/L)for 96 h to evaluate the effect of nitrite stress on the ubiquitin-proteasome system(UPS)in the liver.The expression of genes,the content of ubiquitinated protein,histological alterations of liver,and the levels of serum biochemical parameters were investigated at 12,24,48 and 96 h after exposure,respectively.Compared to the control group,serum cortisol level was significantly elevated at 12 h after exposure in a dose-dependent manner.Meanwhile,the significant rising in serum cortisol level was detected at 24 and 48 h.Serum levels of alanine aminotransferase and aspartate aminotransferase were significantly decreased at 96 h.The mRNA levels of nrf2 and hsp70 increased significantly at 12 h after exposure(20.0 mg/L).Nitrite exposure of 20.0 mg/L caused the significant elevations in mRNA expressions of ub,chip,psmc1,and psma2 as well as in ubiquitinated protein content at 24 h.Hepatic vacuolization was observed in the liver of grass carp at 96 h after exposure to the concentration of nitrite(20.0 mg/L).These results suggested that the exposure of nitrite enhanced the activity of UPS as well as caused stress and histological damage of liver in C.idellus.
引文
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[24]Michael M I,Hilmy A M,El-Domiaty N A,et al.Serum transaminases activity and histopathological changes in Clarias lazera chronically exposed to nitrite [J].Comp Biochem Phys C,1987,86(2):255-262.
[25]Long M,Lin W,Hou J,et al.Dietary supplementation with selenium yeast and tea polyphenols improve growth performance and nitrite tolerance of Wuchang bream(Megalobrama amblycephala)[J].Fish Shellfish Immunol,2017,68:74-83.
[26]Saraste A,Pulkki K.Morphologic and biochemical hallmarks of apoptosis [J].Cardiovasc Res,2000,45(3):528-537.
[27]Zhang X,Yang F,Zhang X,et al.Induction of hepatic enzymes and oxidative stress in Chinese rare minnow(Gobiocypris rarus)exposed to waterborne hexabromocyclododecane(HBCDD)[J].Aquat Toxicol,2008,86(1):4-11.
[2]Thomas S,Wolf D H.Wolf D H.The ubiquitin-proteasome-system [J].Biochim Biophysi Acta,2014,1843(1):1.
[3]Ciechanover A.The ubiquitin-proteasome proteolytic pathway [J].Cell,1994,79(1):13-21.
[4]Itoh K,Mimura J,Yamamoto M.Discovery of the negative regulator of Nrf2,Keap1:a historical overview [J].Antioxid Redox Signal,2010,13(11):1665-1678.
[5]Arndt V,Rogon C,H?hfeld J.To be,or not to be — molecular chaperones in protein degradation [J].Cell Mol Life Sci,2007,64(19-20):2525-2541.
[6]Todgham A E,Crombie T A,Hofmann G E.The effect of temperature adaptation on the ubiquitin-proteasome pathway in notothenioid fishes [J].J Exp Biol,2017,220:369-378
[7]Fang N N,Ng A H M,Vivien M,et al.Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins [J].Nat Cell Biol,2011,13(11):1344-1352.
[8]叶俊,肖琛,尹晓燕,等.亚硝态氮胁迫对草鱼非特异性免疫性能的影响[J].淡水渔业,2013,43(4):40-44.
[9]Xiao C,Liu Z,Li D,et al.Acute nitrite exposure alters the metabolism of thyroid hormones in grass carp(Ctenopharyngodon idellus)[J].Chemosphere,2017,186:798-804.
[10]肖克宇.水产动物免疫与应用[M].北京:科学出版社,2007:182-186.
[11]林艳,缪凌鸿,盘文静,等.亚硝酸盐氮胁迫对鳙血液生化指标以及组织HSP70 mRNA表达水平的影响[J].水产学报,2017,41(8):1192-1202.
[12]Jia R,Han C,Lei J L,et al.Effects of nitrite exposure on haematological parameters,oxidative stress and apoptosis in juvenile turbot(Scophthalmus maximus)[J].Aquat Toxicol,2015,169(7):1-9.
[13]Ciji A,Sahu N P,Pal A K,et al.Alterations in serum electrolytes,antioxidative enzymes and haematological parameters of Labeo rohita on short-term exposure to sublethal dose of nitrite [J].Fish Physiol Biochem,2012,38(5):1355-1365.
[14]Park I S,Lee J,Hur J W,et al.Acute toxicity and sublethal effects of nitrite on selected hematological parameters and tissues in dark-banded rockfish,Sebastes inermis [J].J World Aquacult Soc,2007,38(2):188-199.
[15]强俊,杨弘,王辉,等.急性温度应激对吉富品系尼罗罗非鱼(Oreochromis niloticus)幼鱼生化指标和肝脏HSP70 mRNA表达的影响[J].海洋与湖沼,2012,43(5):943-953.
[16]Lushchak V I.Environmentally induced oxidative stress in aquatic animals [J].Aquat Toxicol,2011,101(1):13-30.
[17]Benarroch E E.Heat shock proteins:multiple neuroprotective functions and implications for neurologic disease [J].Neurology,2011,76(7):660-667.
[18]明建华,谢骏,刘波,等.团头鲂HSP70 cDNA的克隆、序列分析以及热应激对其mRNA表达的影响[J].中国水产科学,2009,16(5):635-648.
[19]Osburn W O,Kensler T W.Nrf2 signaling:an adaptive response pathway for protection against environmental toxic insults [J].Mutat Res,2008,659(1-2):31-39.
[20]Zheng J L,Yuan S S,Wu C W,et al.Acute exposure to waterborne cadmium induced oxidative stress and immunotoxicity in the brain,ovary and liver of zebrafish(Danio rerio)[J].Aquat Toxicol,2016,180:36-44.
[21]叶元土,蔡春芳,许凡,等.氧化鱼油对草鱼肠道黏膜抗氧化应激通路基因表达水平的影响[J].水生生物学报,2016,40(4):758-766.
[22]Tang C H,Leu M Y,Shao K,et al.Short-term effects of thermal stress on the responses of branchial protein quality control and osmoregulation in a reef-associated fish,Chromis viridis [J].Zool Stud,2014,53:21-30.
[23]Rodrigues S,Antunes S C,Nunes B,et al.Histological alterations in gills and liver of rainbow trout(Oncorhynchus mykiss)after exposure to the antibiotic oxytetracycline [J].Environ Toxicol Phar,2017,53:164-176.
[24]Michael M I,Hilmy A M,El-Domiaty N A,et al.Serum transaminases activity and histopathological changes in Clarias lazera chronically exposed to nitrite [J].Comp Biochem Phys C,1987,86(2):255-262.
[25]Long M,Lin W,Hou J,et al.Dietary supplementation with selenium yeast and tea polyphenols improve growth performance and nitrite tolerance of Wuchang bream(Megalobrama amblycephala)[J].Fish Shellfish Immunol,2017,68:74-83.
[26]Saraste A,Pulkki K.Morphologic and biochemical hallmarks of apoptosis [J].Cardiovasc Res,2000,45(3):528-537.
[27]Zhang X,Yang F,Zhang X,et al.Induction of hepatic enzymes and oxidative stress in Chinese rare minnow(Gobiocypris rarus)exposed to waterborne hexabromocyclododecane(HBCDD)[J].Aquat Toxicol,2008,86(1):4-11.