α-酮戊二酸对慢性氨氮胁迫下草鱼肾Rh糖蛋白、热休克蛋白70和过氧化物酶增殖物激活受体γmRNA表达的影响
|
摘要
为探讨饲粮中添加α-酮戊二酸(α-KG)对慢性氨氮胁迫下草鱼(Ctenopharyngodonidellus)肾Rh糖蛋白、热休克蛋白70(HSP70)和过氧化物酶增殖物激活受体γ(PPARγ)mRNA表达的影响,选取初始体重为(24.79±0.11)g的草鱼270尾,随机分成3组:C组(对照组,养于曝气后自来水中并饲喂基础饲粮)、T1组(氨氮组,养于18.37 mg/L的氨氮水中并饲喂基础饲粮)和T2组(α-KG组,养于18.37 mg/L的氨氮水中并饲喂0.75%α-KG饲粮)。氨氮胁迫后1、7、42 d进行采样,测定血氨含量及肾Rhag、Rhcg1、Rhcg2、HSP70和PPARγ的mRNA表达量。结果表明:氨氮胁迫下第1天,T1组血氨含量较对照组显著升高了21%(P<0.05),而日粮α-KG的添加能够有效缓解血氨含量的升高。肾Rhag、Rhcg1和HSP70 mRNA表达量在氨氮胁迫下出现显著差异。其中,氨氮胁迫后1 d,T1组Rhag mRNA表达量较对照组升高了348%(P<0.05),而α-KG的添加能够缓解该现象。氨氮胁迫7 d后,T1组Rhcg1 mRNA表达量与对照组无显著差异,T2组Rhcg1 mRNA的表达量较T1组显著降低了217%(P<0.05)。氨氮胁迫42 d后,T1组和T2组Rhag和Rhcg1 mRNA的表达量均较对照组显著升高(增量分别为405%和833%、59%和48%)(P<0.05)。此外,与T1组相比,T2组肾中HSP70的mRNA表达量显著降低了603%(P<0.05)。而氨氮胁迫7 d后,T1组和T2组HSP70 mRNA表达量较对照组显著降低,且减量分别为408%和404%(P<0.05)。综上所述,饲粮α-KG的添加能够有效缓解前期草鱼血氨含量的升高,并调节氨氮胁迫下Rhag、Rhcg1和HSP70的mRNA表达量,有利于草鱼应对慢性氨氮胁迫。
The objective of this study was to determine the effects of dietary α-ketoglutarate(α-KG) on rhesus glycoproteins, heat shock protein 70(HSP70) and peroxisome proliferator aetivated receptor γ(PPARγ)mRNA expressions of grass carp(Ctenopharyngodonidellus) under chronic ammonia exposure.270 grass carps[initial weight,(24.79 ± 0.11)g] were randomly assigned to three groups:C group,exposed to tap water and fed basic diet;T1 group,exposed to 18.37 mg/L ammonia water and fed basic diet;T2 group,exposed to 18.37 mg/L ammonia water and fed 0.75% α-KG added diet. Grass carps were sampled on day 1,7 and 42,and then the plasma ammonia content and kidney Rhag,Rhcg1,Rhcg2,HSP70,PPARγ mRNA expressions were measured.Results showed that the ammonia level in the T1 group was significantly higher than that in the control group by21% on day 1(P < 0.05),and dietary α-KG alleviated plasma ammonia increased.The kidney Rhag,Rhcg1 and HSP70 mRNA expression showed a significance difference among groups under ammonia exposure.On day 1,the Rhag mRNA expression of T1 group was significant increased 348%(P < 0.05) under ammonia exposure,and α-KG supplement could alleviate this phenomenon. On day 7,the Rhcg1 mRNA expression of C and T1 groups were no significant difference,and the Rhcg1 mRNA expression of T2 group was lower than that in the T1 group by 217%(P < 0.05).On day 42,the mRNA expression of Rhag and Rhcg1 were significant increased under ammonia exposure(the increments were 405% and 833%,59%and 48% respectively)(P < 0.05).Besides,the expression of HSP70 mRNA in T2 group was decreased than that in T1 group by 603%(P < 0.05).On day 7,The expression of HSP70 mRNA in T1 group and T2 group were significantly lower than that in the control group,and the reduction were 408% and 404%,respectively(P < 0.05).In conclusion,dietary α-KG could alleviate the increase of plasma ammonia content and adjust the mRNA expression of Rhag,Rhcg1 and HSP70 under ammonia exposure,which may be beneficial for grass carp to adapt choric ammonia exposure.
The objective of this study was to determine the effects of dietary α-ketoglutarate(α-KG) on rhesus glycoproteins, heat shock protein 70(HSP70) and peroxisome proliferator aetivated receptor γ(PPARγ)mRNA expressions of grass carp(Ctenopharyngodonidellus) under chronic ammonia exposure.270 grass carps[initial weight,(24.79 ± 0.11)g] were randomly assigned to three groups:C group,exposed to tap water and fed basic diet;T1 group,exposed to 18.37 mg/L ammonia water and fed basic diet;T2 group,exposed to 18.37 mg/L ammonia water and fed 0.75% α-KG added diet. Grass carps were sampled on day 1,7 and 42,and then the plasma ammonia content and kidney Rhag,Rhcg1,Rhcg2,HSP70,PPARγ mRNA expressions were measured.Results showed that the ammonia level in the T1 group was significantly higher than that in the control group by21% on day 1(P < 0.05),and dietary α-KG alleviated plasma ammonia increased.The kidney Rhag,Rhcg1 and HSP70 mRNA expression showed a significance difference among groups under ammonia exposure.On day 1,the Rhag mRNA expression of T1 group was significant increased 348%(P < 0.05) under ammonia exposure,and α-KG supplement could alleviate this phenomenon. On day 7,the Rhcg1 mRNA expression of C and T1 groups were no significant difference,and the Rhcg1 mRNA expression of T2 group was lower than that in the T1 group by 217%(P < 0.05).On day 42,the mRNA expression of Rhag and Rhcg1 were significant increased under ammonia exposure(the increments were 405% and 833%,59%and 48% respectively)(P < 0.05).Besides,the expression of HSP70 mRNA in T2 group was decreased than that in T1 group by 603%(P < 0.05).On day 7,The expression of HSP70 mRNA in T1 group and T2 group were significantly lower than that in the control group,and the reduction were 408% and 404%,respectively(P < 0.05).In conclusion,dietary α-KG could alleviate the increase of plasma ammonia content and adjust the mRNA expression of Rhag,Rhcg1 and HSP70 under ammonia exposure,which may be beneficial for grass carp to adapt choric ammonia exposure.
引文
[1]陈亮,梁旭方,瞿春梅,等.草鱼过氧化物酶体增殖物激活受体(PPAR)基因c DNA序列的克隆及其组成型表达[J].暨南大学学报:自然科学与医学版,2011,32(1):80~87.
[2]郭珲.氨转运体Rhbg和Rhcg在低蛋白饮食大鼠肾脏的表达和调控:[博士学位论文][D].太原:山西医科大学,2008.
[3]胡泉舟,侯永清,丁斌鹰,等.α-酮戊二酸对仔猪小肠组织学形态与功能的影响[J].动物营养学报,2008,20(6):662~667.
[4]胡泉舟.α-酮戊二酸对断奶仔猪生长性能和肠道功能的影响:[硕士学位论文][D].武汉:武汉工业学院,2008.
[5]刘坚,侯永清,丁斌鹰,等.α-酮戊二酸对脂多糖应激仔猪肠黏膜能量代谢的影响[J].动物营养学报,2009,6:892~896.
[6]梁健.α-酮戊二酸对草鱼氨氮胁迫的缓解作用研究:[硕士学位论文][D].长沙:湖南农业大学,2014a.
[7]梁健,赵玉蓉,王红权,等.饲料中添加α-酮戊二酸对草鱼热休克蛋白70和过氧化物酶体增殖物激活受体γm RNA表达的影响[J].动物营养学报,2014b,26(5):1379~1386.
[8]刘美莲.过氧化物酶体增殖物激活受体研究的新进展[J].临床与病理杂志,2001,21(5):413~416.
[9]黎庆,龚诗雁,黎明.慢性氨氮暴露诱发黄颡鱼幼鱼谷氨酰胺积累、氧化损伤及免疫抑制的研究[J].水产学报,2015,39(5):728~734.
[10]强俊,徐跑,何杰,等.氨氮与拥挤胁迫对吉富品系尼罗罗非鱼幼鱼生长和肝脏抗氧化指标的联合影响[J].水产学报,2011,35(12):1837~1848.
[11]宋芳杰,王连生,徐奇友.谷氨酰胺及其前体物对松浦镜鲤组织抗氧化能力及血清生化指标的影响[J].动物营养学报,2016,2:627~634.
[12]唐永凯,贾永义.荧光定量PCR数据处理方法的探讨[J].生物技术,2008,18(3):89~91.
[13]王连生,徐奇友,陈迪,等.不同蛋白质源饲料中添加α-酮戊二酸对杂交鲟幼鱼肝脏谷氨酰胺含量、抗氧化能力及生长激素、胰岛素样生长因子-Ⅰ基因表达的影响[J].动物营养学报,2016,28(12):3917~3924.
[14]王蕾,侯永清,丁斌鹰,等.α-酮戊二酸对脂多糖应激仔猪血浆胰岛素、氨基酸含量及腓肠肌m TOR信号传导途径的影响[J].动物营养学报,2010,6:1724~1729.
[15]魏玉强.α-酮戊二酸对松浦镜鲤氮代谢、抗氧化能力和小肠粘膜形态与功能的影响:[硕士学位论文][D].上海:上海海洋大学,2015.
[16]王文.氨转运体蛋白的研究进展[J].中国医师杂志,2014,16(3):427~429.
[17]余亲平,陈雁群,谢金蝉,等.日粮添加α-酮戊二酸对肉仔鸡生长性能及组织器官发育的影响[J].中国畜牧兽医,2010,10:10~14.
[18]杨震国.热休克蛋白70的生物学功能及应用的研究进展[J].中国饲料,2010,23:11~14.
[19]Berger J,Moller D E.The mechanisms of action of PPARs[J].Annual Review of Medicine,2002,53:409~435.
[20]Braissant O,Mclin V A,Cudalbu C.Ammonia toxicity to the brain[J].Journal of Inherited Metabolic Disease,2013,36(4):595~612.
[21]Braun M H,Steele S L,Ekker M,et al.Nitrogen excretion in developing zebrafish(Danio rerio):a role for Rh proteins and urea transporters[J].American Journal of Physiology Renal Physiology,2009,296(5):F994.
[22]Foss A,Siikavuopio S I,S?ther B S,et al.Effect of chronic ammonia exposure on growth in juvenile Atlantic cod[J].Aquaculture,2004,237(1~4):179~189.
[23]Jacquiersarlin M R,Fuller K,Dinhxuan A T,et al.Protective effects of HSP70 in inflammation[J].Experientia,1994,50(11/12):1031~1038.
[24]Kristensen N B,Jungvid H,Fernández J A,et al.Absorption and metabolism of alpha-ketoglutarate in growing pigs[J].Journal of Animal Physiology&Animal Nutrition,2002,86(7~8):239.
[25]Li S F,Liu H X,Zhang Y B,et al.The protective effects of alpha-ketoacids against oxidative stress on rat spermatozoa in vitro[J].Asian Journal of Andrology,2010,12(2):247~56.
[26]Nakano K,Iwama G K.The 70-k Da heat shock protein response in two intertidal sculpins,Oligocottusmaculosus,and O.snyderi:relationship of hsp70 and thermal tolerance[J].Comp Biochem Physiol A Mol Integr Physiol,2002,133(1):79~94.
[27]Nakada T.,Hoshijima K.,Esaki M,et al.Localization of ammonia transporter Rhcg1 in mitochondrion rich cells of yolk sac,gill and kidney of zebrafish and its ionic strength-dependent expression.Am[J].Physiol,2007,293:R1743~R1753.
[28]Pinto W,Aragao C,Soares F,et al.Growth,stress response and free amino acid levels in Senegalese sole(Soleasenegalensis,Kaup1858)chronically exposed to exogenous ammonia[J].Aquaculture Research,2007,38(11):1198~1204.
[29]Qian J H,Li Z M,Liu J Y,et al.Acute toxicity of ammonia-N and its effect on the immune parameters of Fenneropenaeusmerguiensis[J].Journal of Applied Oceanography,2016,2:211~216.
[30]Schram E,Roques J A C,Kuijk T V,et al.The impact of elevated water ammonia and nitrate concentrations on physiology,growth and feed intake of pikeperch(Sander lucioperca)[J].Aquaculture,2014,420~42195~104.
[31]Sinha A K,Liew H J,Nawata C M,et al.Modulation of Rh glycoproteins,ammonia excretion and Na+fluxes in three freshwater teleosts when exposed chronically to high environmental ammonia[J].Journal of Experimental Biology,2013,216(15):2917~30.
[32]Srivastava P.Roles of heat-shock proteins in innate and adaptive immunity[J].Nature Reviews Immunology,2002,2(3):185~194.
[33]Schram E,Roques J A C,Abbink W,et al.The impact of elevated water ammonia concentration on physiology,growth and feed intake of African catfish(Clariasgariepinus)[J].Aquaculture,2010,306(1):108~115.
[34]Vosper H,Khoudoli G A,Graham T L,et al.Peroxisome proliferator-activated receptor agonists,hyperlipidaemia,and atherosclerosis[J].Pharmacology Therapeutics,2002,95(1):47~62.
[35]Wang HQ,Zhao YR,Jin BT,et al.Effects of dietary alpha-ketoglutarate supplementation on growth and serum biochemical parameters of grass carp(Ctenopharyngodonidella)fingerlings[J].Israeli Journal of Aquaculture-Bamidgeh,2016,68(P10):1239.
[36]Weihrauch D,Wilkie M P,Walsh P J.Ammonia and urea transporters in gills of fish and aquatic crustaceans[J].Journal of Experimental Biology,2009,212(11):1716~30.
[37]W elch W J.The role of heat-shock proteins as molecular chaperones[J].Current Opinion in Cell Biology,1991,3(6):1033~1038.
[38]Yao K,Yin Y,Li X,et al.Alpha-ketoglutarate inhibits glutamine degradation and enhances protein synthesis in intestinal porcine epithelial cells[J].Amino Acids,2012,42(6):2491~2500.
[2]郭珲.氨转运体Rhbg和Rhcg在低蛋白饮食大鼠肾脏的表达和调控:[博士学位论文][D].太原:山西医科大学,2008.
[3]胡泉舟,侯永清,丁斌鹰,等.α-酮戊二酸对仔猪小肠组织学形态与功能的影响[J].动物营养学报,2008,20(6):662~667.
[4]胡泉舟.α-酮戊二酸对断奶仔猪生长性能和肠道功能的影响:[硕士学位论文][D].武汉:武汉工业学院,2008.
[5]刘坚,侯永清,丁斌鹰,等.α-酮戊二酸对脂多糖应激仔猪肠黏膜能量代谢的影响[J].动物营养学报,2009,6:892~896.
[6]梁健.α-酮戊二酸对草鱼氨氮胁迫的缓解作用研究:[硕士学位论文][D].长沙:湖南农业大学,2014a.
[7]梁健,赵玉蓉,王红权,等.饲料中添加α-酮戊二酸对草鱼热休克蛋白70和过氧化物酶体增殖物激活受体γm RNA表达的影响[J].动物营养学报,2014b,26(5):1379~1386.
[8]刘美莲.过氧化物酶体增殖物激活受体研究的新进展[J].临床与病理杂志,2001,21(5):413~416.
[9]黎庆,龚诗雁,黎明.慢性氨氮暴露诱发黄颡鱼幼鱼谷氨酰胺积累、氧化损伤及免疫抑制的研究[J].水产学报,2015,39(5):728~734.
[10]强俊,徐跑,何杰,等.氨氮与拥挤胁迫对吉富品系尼罗罗非鱼幼鱼生长和肝脏抗氧化指标的联合影响[J].水产学报,2011,35(12):1837~1848.
[11]宋芳杰,王连生,徐奇友.谷氨酰胺及其前体物对松浦镜鲤组织抗氧化能力及血清生化指标的影响[J].动物营养学报,2016,2:627~634.
[12]唐永凯,贾永义.荧光定量PCR数据处理方法的探讨[J].生物技术,2008,18(3):89~91.
[13]王连生,徐奇友,陈迪,等.不同蛋白质源饲料中添加α-酮戊二酸对杂交鲟幼鱼肝脏谷氨酰胺含量、抗氧化能力及生长激素、胰岛素样生长因子-Ⅰ基因表达的影响[J].动物营养学报,2016,28(12):3917~3924.
[14]王蕾,侯永清,丁斌鹰,等.α-酮戊二酸对脂多糖应激仔猪血浆胰岛素、氨基酸含量及腓肠肌m TOR信号传导途径的影响[J].动物营养学报,2010,6:1724~1729.
[15]魏玉强.α-酮戊二酸对松浦镜鲤氮代谢、抗氧化能力和小肠粘膜形态与功能的影响:[硕士学位论文][D].上海:上海海洋大学,2015.
[16]王文.氨转运体蛋白的研究进展[J].中国医师杂志,2014,16(3):427~429.
[17]余亲平,陈雁群,谢金蝉,等.日粮添加α-酮戊二酸对肉仔鸡生长性能及组织器官发育的影响[J].中国畜牧兽医,2010,10:10~14.
[18]杨震国.热休克蛋白70的生物学功能及应用的研究进展[J].中国饲料,2010,23:11~14.
[19]Berger J,Moller D E.The mechanisms of action of PPARs[J].Annual Review of Medicine,2002,53:409~435.
[20]Braissant O,Mclin V A,Cudalbu C.Ammonia toxicity to the brain[J].Journal of Inherited Metabolic Disease,2013,36(4):595~612.
[21]Braun M H,Steele S L,Ekker M,et al.Nitrogen excretion in developing zebrafish(Danio rerio):a role for Rh proteins and urea transporters[J].American Journal of Physiology Renal Physiology,2009,296(5):F994.
[22]Foss A,Siikavuopio S I,S?ther B S,et al.Effect of chronic ammonia exposure on growth in juvenile Atlantic cod[J].Aquaculture,2004,237(1~4):179~189.
[23]Jacquiersarlin M R,Fuller K,Dinhxuan A T,et al.Protective effects of HSP70 in inflammation[J].Experientia,1994,50(11/12):1031~1038.
[24]Kristensen N B,Jungvid H,Fernández J A,et al.Absorption and metabolism of alpha-ketoglutarate in growing pigs[J].Journal of Animal Physiology&Animal Nutrition,2002,86(7~8):239.
[25]Li S F,Liu H X,Zhang Y B,et al.The protective effects of alpha-ketoacids against oxidative stress on rat spermatozoa in vitro[J].Asian Journal of Andrology,2010,12(2):247~56.
[26]Nakano K,Iwama G K.The 70-k Da heat shock protein response in two intertidal sculpins,Oligocottusmaculosus,and O.snyderi:relationship of hsp70 and thermal tolerance[J].Comp Biochem Physiol A Mol Integr Physiol,2002,133(1):79~94.
[27]Nakada T.,Hoshijima K.,Esaki M,et al.Localization of ammonia transporter Rhcg1 in mitochondrion rich cells of yolk sac,gill and kidney of zebrafish and its ionic strength-dependent expression.Am[J].Physiol,2007,293:R1743~R1753.
[28]Pinto W,Aragao C,Soares F,et al.Growth,stress response and free amino acid levels in Senegalese sole(Soleasenegalensis,Kaup1858)chronically exposed to exogenous ammonia[J].Aquaculture Research,2007,38(11):1198~1204.
[29]Qian J H,Li Z M,Liu J Y,et al.Acute toxicity of ammonia-N and its effect on the immune parameters of Fenneropenaeusmerguiensis[J].Journal of Applied Oceanography,2016,2:211~216.
[30]Schram E,Roques J A C,Kuijk T V,et al.The impact of elevated water ammonia and nitrate concentrations on physiology,growth and feed intake of pikeperch(Sander lucioperca)[J].Aquaculture,2014,420~42195~104.
[31]Sinha A K,Liew H J,Nawata C M,et al.Modulation of Rh glycoproteins,ammonia excretion and Na+fluxes in three freshwater teleosts when exposed chronically to high environmental ammonia[J].Journal of Experimental Biology,2013,216(15):2917~30.
[32]Srivastava P.Roles of heat-shock proteins in innate and adaptive immunity[J].Nature Reviews Immunology,2002,2(3):185~194.
[33]Schram E,Roques J A C,Abbink W,et al.The impact of elevated water ammonia concentration on physiology,growth and feed intake of African catfish(Clariasgariepinus)[J].Aquaculture,2010,306(1):108~115.
[34]Vosper H,Khoudoli G A,Graham T L,et al.Peroxisome proliferator-activated receptor agonists,hyperlipidaemia,and atherosclerosis[J].Pharmacology Therapeutics,2002,95(1):47~62.
[35]Wang HQ,Zhao YR,Jin BT,et al.Effects of dietary alpha-ketoglutarate supplementation on growth and serum biochemical parameters of grass carp(Ctenopharyngodonidella)fingerlings[J].Israeli Journal of Aquaculture-Bamidgeh,2016,68(P10):1239.
[36]Weihrauch D,Wilkie M P,Walsh P J.Ammonia and urea transporters in gills of fish and aquatic crustaceans[J].Journal of Experimental Biology,2009,212(11):1716~30.
[37]W elch W J.The role of heat-shock proteins as molecular chaperones[J].Current Opinion in Cell Biology,1991,3(6):1033~1038.
[38]Yao K,Yin Y,Li X,et al.Alpha-ketoglutarate inhibits glutamine degradation and enhances protein synthesis in intestinal porcine epithelial cells[J].Amino Acids,2012,42(6):2491~2500.