高温对淮南麻黄鸡血脑屏障的影响
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摘要
为探究高温对淮南麻黄鸡血脑屏障的影响,将120羽107日龄淮南麻黄鸡随机均分为四组:常温对照组(Ⅰ组)、常温注射组(Ⅱ组)、高温对照组(Ⅲ组)、高温注射组(Ⅳ组)。Ⅰ、Ⅱ组于常温下饲养(21~23℃、RH60%~70%),Ⅲ、Ⅳ组每天高温处理3h(34~37℃、RH 45%~50%),试验持续2周。试验第7、14天,每组随机抽取5羽鸡,Ⅱ、Ⅳ组鸡一次静脉注射γ-氨基丁酸(GABA)(每千克体重8.87mg),Ⅰ、Ⅲ组鸡注射同等剂量的生理盐水,注射0.5h后断头取脑,冰浴上迅速采集脑组织。使用ELISA法分别检测第7、14天四组鸡脑组织中的GABA含量;采用电镜技术观察第14天常温对照组和高温对照组中鸡脑组织的血脑屏障超微结构,并用qRT-PCR技术和Western blot技术检测其脑组织中紧密连接蛋白ZO-1和Occludin的mRNA及蛋白质的表达量。结果显示:试验第7天,Ⅰ、Ⅱ组鸡脑组织中GABA含量无显著差异(P>0.05),Ⅳ组鸡脑组织中GABA含量极显著高于Ⅲ组(P<0.01);试验第14天,Ⅰ、Ⅱ组间鸡脑组织中GABA含量无显著差异(P>0.05),Ⅳ组鸡脑组织中GABA含量高于Ⅲ组,但差异不显著(P>0.05)。与常温对照组相比,高温对照组鸡的血脑屏障结构受到破坏,电镜下观察到大脑皮层血管内皮细胞线粒体嵴有的断裂、有的空泡变性,血管内皮细胞间紧密连接间隙增宽,内皮细胞外基膜增厚、结构疏松;高温对照组鸡脑组织ZO-1的mRNA相对转录量极显著低于常温对照组(P<0.01),Occludin的mRNA相对转录量显著低于常温对照组(P<0.05);高温对照组鸡脑组织ZO-1和Occludin蛋白的相对表达量极显著低于常温对照组(P<0.01)。高温破坏了鸡的血脑屏障,脑组织中ZO-1和Occludin表达下降可能是其原因之一。
The aim of the present study was to explore the impact of high temperature on Huainan partridge chickens' blood-brain barrier.One hundred and twenty 107-day old Huainan partridge chickens were randomly divided into four groups:chickens in groupⅠandⅡ were raised at normal room temperature(21-23 ℃,RH 60%-70%),which in GroupⅢandⅣsubjected to hyperthermia(34-37 ℃,RH 45%-50%)for 3hours daily.The treatment lasted for two weeks.Five chickens were randomly selected from each group on day 7and 14,respectively,chickens in group Ⅱ and Ⅳ were received a single intravenous dose of Gamma-aminobutyric acid(GABA)(8.87mg·kg-1 body weight),chickens in group Ⅰ and Ⅲ received same dose of saline,brain tissue samples were collected at time 0.5hand analyzed GABA using ELISA assay.The braintissue samples were collected on day 14,and the ultrastructure of the blood brain barrier were detected using transmission electron microscopy(TEM),the mRNA and protein expression of ZO-1and Occludin in the brain tissues were detected by qRT-PCR and Western blot.The results showed that,On day 7and 14,no significant differences of brain GABA concentration were found between group Ⅰ and Ⅱ(P>0.05).Brain GABA concentration of chicks in Group Ⅳwere higher than that in group Ⅲ on day 7and 14,and the differences were very significant on day 7(P<0.01).Compared with normal temperature,high temperature destroyed the bloodbrain barrier structure of chickens.Mitochondrial cristae of Cerebral cortical capillary endothelial cells were damaged,some broken,some degeneration.Capillary endothelial cell tight junctions gap widened.Microvascular basement membrane became loose and thick.The mRNA transcription of ZO-1and Occludin were very significantly(P<0.01)and significantly lower(P<0.05)than those in normal temperature group,respectively.The protein relative expression of ZO-1and Occludin were very significantly(P<0.01)lower on days 14 than those in normal temperature group.High temperature destroyed the chicken blood-brain barrier,ZO-1and Occludin down-regulation may be one of the mediators of it.
The aim of the present study was to explore the impact of high temperature on Huainan partridge chickens' blood-brain barrier.One hundred and twenty 107-day old Huainan partridge chickens were randomly divided into four groups:chickens in groupⅠandⅡ were raised at normal room temperature(21-23 ℃,RH 60%-70%),which in GroupⅢandⅣsubjected to hyperthermia(34-37 ℃,RH 45%-50%)for 3hours daily.The treatment lasted for two weeks.Five chickens were randomly selected from each group on day 7and 14,respectively,chickens in group Ⅱ and Ⅳ were received a single intravenous dose of Gamma-aminobutyric acid(GABA)(8.87mg·kg-1 body weight),chickens in group Ⅰ and Ⅲ received same dose of saline,brain tissue samples were collected at time 0.5hand analyzed GABA using ELISA assay.The braintissue samples were collected on day 14,and the ultrastructure of the blood brain barrier were detected using transmission electron microscopy(TEM),the mRNA and protein expression of ZO-1and Occludin in the brain tissues were detected by qRT-PCR and Western blot.The results showed that,On day 7and 14,no significant differences of brain GABA concentration were found between group Ⅰ and Ⅱ(P>0.05).Brain GABA concentration of chicks in Group Ⅳwere higher than that in group Ⅲ on day 7and 14,and the differences were very significant on day 7(P<0.01).Compared with normal temperature,high temperature destroyed the bloodbrain barrier structure of chickens.Mitochondrial cristae of Cerebral cortical capillary endothelial cells were damaged,some broken,some degeneration.Capillary endothelial cell tight junctions gap widened.Microvascular basement membrane became loose and thick.The mRNA transcription of ZO-1and Occludin were very significantly(P<0.01)and significantly lower(P<0.05)than those in normal temperature group,respectively.The protein relative expression of ZO-1and Occludin were very significantly(P<0.01)lower on days 14 than those in normal temperature group.High temperature destroyed the chicken blood-brain barrier,ZO-1and Occludin down-regulation may be one of the mediators of it.
引文
[1]吴凡,谭青松.γ-氨基丁酸在动物营养中的研究进展[J].饲料研究,2015(2):61-65.WU F,TAN Q S.Advances ofγ-aminobutyric acid in animal nutrition[J].Feed Research,2015(2):61-65.(in Chinese)
[2]白东鲁,陈凯先.药物化学进展[M].北京:化学工业出版社,2005.BAI D L,CHEN K X.Advances in pharmaceutical chemistry[M].Beijing:Chemical Industry Press,2005.(in Chinese)
[3]梁臣,陈忠.γ-氨基丁酸及其受体功能的研究与应用现状[J].动物医学进展,2015,36(4):108-112.LIANG C,CHEN Z.Research and application status on physiologic functions ofγ-aminobutyric acid and its receptors[J].Progress in Veterinary Medicine,2015,36(4):108-112.(in Chinese)
[4]HU H,BAI X,SHAH A A,et al.Dietary supplementation with glutamine andγ-aminobutyric acid improves growth performance and serum parameters in22-to 35-day-old broilers exposed to hot environment[J].J Anim Physiol Anim Nutr,2016,100(2):361-370.
[5]CHEN Z,XIE J,WANG B,et al.Effect ofγ-aminobutyric acid on digestive enzymes,absorption function,and immune function of intestinal mucosa in heat-stressed chicken[J].Poult Sci,2014,93(10):2490-2500.
[6]CHENG J B,BU D P,WANG J Q,et al.Effects of rumen-protectedγ-aminobutyric acid on performance and nutrient digestibility in heat-stressed dairy cows[J].J Dairy Sci,2014,97(9):5599-5607.
[7]HSU C C,CHEN S H,LIN C H,et al.Human recombinant factor VIIa may improve heat intolerance in mice by attenuating hypothalamic neuronal apoptosis and damage[J].Apoptosis,2014,19(10):1484-1496.
[8]MICHEL V,PEINNEQUIN A,ALONSO A,et al.Decreased heat tolerance is associated with hypothalamo-pituitary-adrenocortical axis impairment[J].Neuroscience,2007,147(2):522-531.
[9]罗学港.神经科学基础[M].长沙:中南大学出版社,2016.LUO X G.Basic neuroscience[M].Changsha:Central South University Press,2016.(in Chinese)
[10]MELN L C,MAGUIRE J.GABAergic regulation of the HPA and HPG axes and the impact of stress on reproductive function[J].J Steroid Biochem Mol Biol,2015,160:196-203.
[11]徐秋良,张庆莉,唐国盘,等.γ氨基丁酸对高温环境下生长肥育猪生产性能及激素水平的影响[J].家畜生态学报,2009,30(6):28-32.XU Q L,ZHANG Q L,TANG G P,et al.Effect ofγ-aminobutyric acid on performance and hormones of finishing pig in heat stress environment[J].Acta Ecologiae Animalis Domastici,2009,30(6):28-32.(in Chinese)
[12]胡家澄,邹晓庭,赵文静,等.γ-氨基丁酸对生长肥育猪生长性能、血清生化指标及HPA、HPT轴激素分泌的影响[J].动物营养学报,2009,21(2):226-231.HU J C,ZOU X T,ZHAO W J,et al.Effect ofγ-aminobutyric acid on growth performance,serum biochemical indices and hormones of HPA and HPT axis in growing-finishing swine[J].Journal of Animal Nutrition,2009,21(2):226-231.(in Chinese)
[13]徐同锏,钟刚,王希春,等.脂肪包被γ-氨基丁酸对持续热应激奶牛HPA轴和部分免疫指标的影响[J].南京农业大学学报,2014,37(1):127-132.XU T J,ZHONG G,WANG X C,et al.Effects of lipid coatedγ-aminobutyric acid on HPA axis and some immune indicators of cows in heat stress[J].Journal of Nanjing Agricultural University,2014,37(1):127-132.(in Chinese)
[14]HAWKER D D,SILVERMAN R B.Synthesis and evaluation of novel heteroaromatic substrates of GABA aminotransferase[J].Bioorg Med Chem,2012,20(19):5763-5773.
[15]LE H V,HAWKER D D,WU R,et al.Design and mechanism of tetrahydrothiophene-basedγ-aminobutyric acid aminotransferase inactivators[J].J Am Chem Soc,2015,137(13):4525-4533.
[16]张礼.γ-氨基丁酸对热应激肉鸭生产性能的影响[D].泰安:山东农业大学,2010.ZHANG L.Effects ofγ-aminobutyric acid on the performance of meat ducks which were suffered from heat stress[D].Tai’an:Shandong Agricultural University,2010.(in Chinese)
[17]SINHA R K.Study of changes in some pathophysiological stress markers in different age groups of an animal model of acute and chronic heat stress[J].Iran Biomed J,2007,11(2):101-111.
[18]MURESANU D F,SHARMA A,SHARMA H S.Diabetes aggravates heat stress-induced blood-brain barrier breakdown,reduction in cerebral blood flow,edema formation,and brain pathology[J].Ann N Y Acad Sci,2010,1199:15-26.
[19]MURESANU D F,SHARMA H S.Chronic hypertension aggravates heat stress-induced cognitive dysfunction and brain pathology:an experimental study in the rat,using growth hormone therapy for possible neuroprotection[J].Ann N Y Acad Sci,2007,1122:1-22.
[20]斯托凯.禽类生理学[M].《禽类生理学》翻译组,译.北京:科学出版社,1982.STURKIE P D.Avian physiology[M].Translation Group of Avian Physiology,Trans.Beijing:Science Press,1982.(in Chinese)
[21]杨倩.动物组织学与胚胎学[M].北京:中国农业大学出版社,2008.YANG Q.Histology and embryology of animal[M].Beijing:China Agricultural University Press,2008.(in Chinese)
[22]张春丽,丘增云,孟强.高温通过下调密封蛋白-1表达加重局灶性脑缺血再灌注大鼠脑损伤[J].国际脑血管病杂志,2013,21(10):775-780.ZHANG C L,QIU Z Y,MENG Q.Hyperthermia aggravates focal cerebral ischemia-reperfusion injury in rats by down-regulating claudin-1expression[J].International Journal of Cerebrovascular Diseases,2013,21(10):775-780.(in Chinese)
[23]CHAO C M,CHENG B C,CHEN C Y,et al.Proteomic analysis of hypothalamic injury in heatstroke rats[J].Proteomics,2015,15(11):1921-1934.
[24]SHARMA H S,HOOPES P J.Hyperthermia induced pathophysiology of the central nervous system[J].Int J Hyperthermia,2003,19(3):325-354.
[25]ABBOTT N J,PATABENDIGE A A K,DOLMAN D E M,et al.Structure and function of the bloodbrain barrier[J].Neurobiol Dis,2010,37(1):13-25.
[26]KNYCH H K,STEINMETZ S J,MCKEMIE D S.Endogenous concentrations,pharmacokinetics,and selected pharmacodynamic effects of a single dose of exogenous GABA in horses[J].J Vet Pharmacol Therap,2015,38(2):113-122.
[27]VAN GELDER N M,ELLIOTT K A C.Disposition ofγ-aminobutyric acid administered to mammals[J].J Neurochem,1958,3(2):139-143.
[28]韩雁冰,王学锋,李增友,等.静脉滴注GABA联合QPEEG评价血脑屏障完整性初探[J].癫癎与神经电生理学杂志,2011,20(1):39-43.HAN Y B,WANG X F,LI Z Y,et al.Intravenous GABA in combination with QPEEG to assess the integrity of BBB[J].Journal of Epileptology and Electroneurophysiology(China),2011,20(1):39-43.(in Chinese)
[29]邓孔昭,黄如训.血脑屏障[M].北京:人民卫生出版社,1984.DENG K Z,HUANG R X.Blood-brain barrier[M].Beijing:People’s Medical Publishing House,1984.(in Chinese)
[30]姬从花.组织蛋白酶L及高温对体外血脑屏障紧密连接蛋白claudin-1的影响[D].昆明:昆明医科大学,2014.JI C H.Effect of cathepsin L and hyperthermia on tight junctions between endothelial cells of the bloodbrain barrier model in vitro[D].Kunming:Kunming Medical University,2014.(in Chinese)
[31]DENKER B M,NIGAM S K.Molecular structure and assembly of the tight junction[J].Am J Physiol,1998,274(1):F1-F9.
[32]陈祎招,徐如祥,黄柒金,等.高温对血脑屏障内皮细胞紧密连接的影响[J].第一军医大学学报,2003,23(1):21-24.CHEN Y Z,XU R X,HUANG Q J,et al.Effect of hyperthermia on tight junctions between endothelial cells of the blood-brain barrier model in vitro[J].Journal of First Military Medical University,2003,23(1):21-24.(in Chinese)
[33]ZHAO J Y,HAO J H,FEI X,et al.Isoflurane inhibits occludin expression via up-regulation of hypoxia-inducible factor 1α[J].Brain Res,2014,1562:1-10.
[34]XU R F,FENG X Y,XIE X,et al.HIV-1Tat protein increases the permeability of brain endothelial cells by both inhibiting occludin expression and cleaving occludin via matrix metalloproteinase-9[J].Brain Res,2012,1436:13-19.
[2]白东鲁,陈凯先.药物化学进展[M].北京:化学工业出版社,2005.BAI D L,CHEN K X.Advances in pharmaceutical chemistry[M].Beijing:Chemical Industry Press,2005.(in Chinese)
[3]梁臣,陈忠.γ-氨基丁酸及其受体功能的研究与应用现状[J].动物医学进展,2015,36(4):108-112.LIANG C,CHEN Z.Research and application status on physiologic functions ofγ-aminobutyric acid and its receptors[J].Progress in Veterinary Medicine,2015,36(4):108-112.(in Chinese)
[4]HU H,BAI X,SHAH A A,et al.Dietary supplementation with glutamine andγ-aminobutyric acid improves growth performance and serum parameters in22-to 35-day-old broilers exposed to hot environment[J].J Anim Physiol Anim Nutr,2016,100(2):361-370.
[5]CHEN Z,XIE J,WANG B,et al.Effect ofγ-aminobutyric acid on digestive enzymes,absorption function,and immune function of intestinal mucosa in heat-stressed chicken[J].Poult Sci,2014,93(10):2490-2500.
[6]CHENG J B,BU D P,WANG J Q,et al.Effects of rumen-protectedγ-aminobutyric acid on performance and nutrient digestibility in heat-stressed dairy cows[J].J Dairy Sci,2014,97(9):5599-5607.
[7]HSU C C,CHEN S H,LIN C H,et al.Human recombinant factor VIIa may improve heat intolerance in mice by attenuating hypothalamic neuronal apoptosis and damage[J].Apoptosis,2014,19(10):1484-1496.
[8]MICHEL V,PEINNEQUIN A,ALONSO A,et al.Decreased heat tolerance is associated with hypothalamo-pituitary-adrenocortical axis impairment[J].Neuroscience,2007,147(2):522-531.
[9]罗学港.神经科学基础[M].长沙:中南大学出版社,2016.LUO X G.Basic neuroscience[M].Changsha:Central South University Press,2016.(in Chinese)
[10]MELN L C,MAGUIRE J.GABAergic regulation of the HPA and HPG axes and the impact of stress on reproductive function[J].J Steroid Biochem Mol Biol,2015,160:196-203.
[11]徐秋良,张庆莉,唐国盘,等.γ氨基丁酸对高温环境下生长肥育猪生产性能及激素水平的影响[J].家畜生态学报,2009,30(6):28-32.XU Q L,ZHANG Q L,TANG G P,et al.Effect ofγ-aminobutyric acid on performance and hormones of finishing pig in heat stress environment[J].Acta Ecologiae Animalis Domastici,2009,30(6):28-32.(in Chinese)
[12]胡家澄,邹晓庭,赵文静,等.γ-氨基丁酸对生长肥育猪生长性能、血清生化指标及HPA、HPT轴激素分泌的影响[J].动物营养学报,2009,21(2):226-231.HU J C,ZOU X T,ZHAO W J,et al.Effect ofγ-aminobutyric acid on growth performance,serum biochemical indices and hormones of HPA and HPT axis in growing-finishing swine[J].Journal of Animal Nutrition,2009,21(2):226-231.(in Chinese)
[13]徐同锏,钟刚,王希春,等.脂肪包被γ-氨基丁酸对持续热应激奶牛HPA轴和部分免疫指标的影响[J].南京农业大学学报,2014,37(1):127-132.XU T J,ZHONG G,WANG X C,et al.Effects of lipid coatedγ-aminobutyric acid on HPA axis and some immune indicators of cows in heat stress[J].Journal of Nanjing Agricultural University,2014,37(1):127-132.(in Chinese)
[14]HAWKER D D,SILVERMAN R B.Synthesis and evaluation of novel heteroaromatic substrates of GABA aminotransferase[J].Bioorg Med Chem,2012,20(19):5763-5773.
[15]LE H V,HAWKER D D,WU R,et al.Design and mechanism of tetrahydrothiophene-basedγ-aminobutyric acid aminotransferase inactivators[J].J Am Chem Soc,2015,137(13):4525-4533.
[16]张礼.γ-氨基丁酸对热应激肉鸭生产性能的影响[D].泰安:山东农业大学,2010.ZHANG L.Effects ofγ-aminobutyric acid on the performance of meat ducks which were suffered from heat stress[D].Tai’an:Shandong Agricultural University,2010.(in Chinese)
[17]SINHA R K.Study of changes in some pathophysiological stress markers in different age groups of an animal model of acute and chronic heat stress[J].Iran Biomed J,2007,11(2):101-111.
[18]MURESANU D F,SHARMA A,SHARMA H S.Diabetes aggravates heat stress-induced blood-brain barrier breakdown,reduction in cerebral blood flow,edema formation,and brain pathology[J].Ann N Y Acad Sci,2010,1199:15-26.
[19]MURESANU D F,SHARMA H S.Chronic hypertension aggravates heat stress-induced cognitive dysfunction and brain pathology:an experimental study in the rat,using growth hormone therapy for possible neuroprotection[J].Ann N Y Acad Sci,2007,1122:1-22.
[20]斯托凯.禽类生理学[M].《禽类生理学》翻译组,译.北京:科学出版社,1982.STURKIE P D.Avian physiology[M].Translation Group of Avian Physiology,Trans.Beijing:Science Press,1982.(in Chinese)
[21]杨倩.动物组织学与胚胎学[M].北京:中国农业大学出版社,2008.YANG Q.Histology and embryology of animal[M].Beijing:China Agricultural University Press,2008.(in Chinese)
[22]张春丽,丘增云,孟强.高温通过下调密封蛋白-1表达加重局灶性脑缺血再灌注大鼠脑损伤[J].国际脑血管病杂志,2013,21(10):775-780.ZHANG C L,QIU Z Y,MENG Q.Hyperthermia aggravates focal cerebral ischemia-reperfusion injury in rats by down-regulating claudin-1expression[J].International Journal of Cerebrovascular Diseases,2013,21(10):775-780.(in Chinese)
[23]CHAO C M,CHENG B C,CHEN C Y,et al.Proteomic analysis of hypothalamic injury in heatstroke rats[J].Proteomics,2015,15(11):1921-1934.
[24]SHARMA H S,HOOPES P J.Hyperthermia induced pathophysiology of the central nervous system[J].Int J Hyperthermia,2003,19(3):325-354.
[25]ABBOTT N J,PATABENDIGE A A K,DOLMAN D E M,et al.Structure and function of the bloodbrain barrier[J].Neurobiol Dis,2010,37(1):13-25.
[26]KNYCH H K,STEINMETZ S J,MCKEMIE D S.Endogenous concentrations,pharmacokinetics,and selected pharmacodynamic effects of a single dose of exogenous GABA in horses[J].J Vet Pharmacol Therap,2015,38(2):113-122.
[27]VAN GELDER N M,ELLIOTT K A C.Disposition ofγ-aminobutyric acid administered to mammals[J].J Neurochem,1958,3(2):139-143.
[28]韩雁冰,王学锋,李增友,等.静脉滴注GABA联合QPEEG评价血脑屏障完整性初探[J].癫癎与神经电生理学杂志,2011,20(1):39-43.HAN Y B,WANG X F,LI Z Y,et al.Intravenous GABA in combination with QPEEG to assess the integrity of BBB[J].Journal of Epileptology and Electroneurophysiology(China),2011,20(1):39-43.(in Chinese)
[29]邓孔昭,黄如训.血脑屏障[M].北京:人民卫生出版社,1984.DENG K Z,HUANG R X.Blood-brain barrier[M].Beijing:People’s Medical Publishing House,1984.(in Chinese)
[30]姬从花.组织蛋白酶L及高温对体外血脑屏障紧密连接蛋白claudin-1的影响[D].昆明:昆明医科大学,2014.JI C H.Effect of cathepsin L and hyperthermia on tight junctions between endothelial cells of the bloodbrain barrier model in vitro[D].Kunming:Kunming Medical University,2014.(in Chinese)
[31]DENKER B M,NIGAM S K.Molecular structure and assembly of the tight junction[J].Am J Physiol,1998,274(1):F1-F9.
[32]陈祎招,徐如祥,黄柒金,等.高温对血脑屏障内皮细胞紧密连接的影响[J].第一军医大学学报,2003,23(1):21-24.CHEN Y Z,XU R X,HUANG Q J,et al.Effect of hyperthermia on tight junctions between endothelial cells of the blood-brain barrier model in vitro[J].Journal of First Military Medical University,2003,23(1):21-24.(in Chinese)
[33]ZHAO J Y,HAO J H,FEI X,et al.Isoflurane inhibits occludin expression via up-regulation of hypoxia-inducible factor 1α[J].Brain Res,2014,1562:1-10.
[34]XU R F,FENG X Y,XIE X,et al.HIV-1Tat protein increases the permeability of brain endothelial cells by both inhibiting occludin expression and cleaving occludin via matrix metalloproteinase-9[J].Brain Res,2012,1436:13-19.