特丁基对苯二酚对氯化镧所致原代培养大鼠大脑皮层神经元氧化损伤的拮抗作用
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摘要
目的探讨特丁基对苯二酚(tertiary butylhydroquinone,tBHQ)对氯化镧所致原代培养大鼠大脑皮层神经元氧化损伤的拮抗作用。方法将出生24 h内清洁级Wistar大鼠的胎鼠大脑皮质神经元进行原代培养,采用神经元特异性烯醇化酶(NSE)免疫细胞荧光法进行神经元的纯度鉴定;将神经元分别暴露于终浓度为0(对照)、0.125、0.25、0.5 mmol/L氯化镧的DMEM培养基中染毒24 h,同时,设40μmol/L tBHQ与相应浓度氯化镧干预组。采用二乙酰二氯荧光素(DCFH-DA)探针测定神经元内活性氧(ROS)的水平,采用Western blot法测定神经元内核因子NF-E2相关因子(nuclear factor erythroid2-related factor,Nrf2)、谷胱甘肽S转移酶(glutathione S transferase,GST)和超氧化物歧化酶2(superoxide dismutase 2,SOD_2)蛋白的表达水平。结果与对照组比较,各浓度氯化镧染毒组原代培养大鼠神经元ROS水平均升高,差异有统计学意义(P<0.05);且随着氯化镧染毒浓度的升高,原代培养大鼠神经元内的ROS水平呈上升趋势。与相同浓度氯化镧组比较,0.25、0.5 mmol/L氯化镧+40μmol/L t BHQ干预组原代培养大鼠神经元的ROS水平均较低,差异有统计学意义(P<0.05)。与对照组相比,各浓度氯化镧组神经元内Nrf2、GST和SOD_2蛋白的表达水平均降低,除0.125 mmol/L氯化镧染毒组外,差异均有统计学意义(P<0.05);且随着氯化镧染毒浓度的升高,原代培养大鼠神经元内Nrf2、GST和SOD_2蛋白的表达水平均呈下降趋势。与相同浓度氯化镧组比较,0.125、0.25、0.5 mmol/L氯化镧+40μmol/L tBHQ干预组原代培养大鼠神经元的Nrf2、GST和SOD_2蛋白的表达水平均增加,除0.25 mmol/L氯化镧+40μmol/L tBHQ干预组SOD_2蛋白的表达水平外,差异有统计学意义(P<0.05)。结论氯化镧可使神经元内ROS水平增加,Nrf2,GST和SOD_2的蛋白表达水平减少,造成神经元发生氧化损伤;而tBHQ干预可拮抗镧所致的ROS水平增加、Nrf2及抗氧化蛋白的表达降低,对神经元具有保护作用。
Objective To explore antagonistic effects of tert-butyl hydroquinone(tBHQ)on oxidative damage of primary cerebral cortical neurons in rats induced by lanthanum chloride(LaCl_3 ).Methods The newborn Wistar rats within 24 hours were collected to performed primary neurons culture.Immunofluorescence(NSE)was used to identify the cultured neurons.Neurons were exposed to 0(control),0.125,0.25 and 0.5 mmol/L LaCl_3 in DMEM medium respectively,and also tBHQ(40μmol/L)+0.125mmol/L LaCl_3 ,tBHQ(40μmol/L)+0.25 mmol/L LaCl_3 and tBHQ(40μmol/L)+0.5 mmol/L LaCl_3 respectively for 24 hours respectively.Diacetyl dichlorofluorescein(DCFH-DA)was used to detect ROS levels of neurons.Western blot was used to measure Nrf2,GST and SOD_2 protein expression levels of neurons.Results Compared with the control group,ROS levels of primary neurons in 0.125,0.25 and 0.5 mmol/L LaCl_3 groups increased significantly(P<0.05),with a dose dependent manner.Compared with the related LaCl_3 exposure groups,ROS levels of primary neurons in 0.25 mmol/L LaCl_3 +40μmol/L tBHQ group and 0.5 mmol/L LaCl_3 +40μmol/L tBHQ group decreased significantly(P<0.05).Compared with the control group,Nrf2,GST and SOD_2 protein expression levels in 0.25 and 0.5 mmol/L LaCl_3 groups decreased significantly(P<0.05),except 0.125 mmol/L LaCl_3 group,and Nrf2,GST and SOD_2 protein expression levels gradually decreased with the increase of LaCl_3 exposure doses.Compared with the related LaCl_3 exposure groups,Nrf2,GST and SOD_2 protein expression levels of primary neurons in 0.125mmol/L LaCl_3 +40μmol/L tBHQ group,0.25 mmol/L LaCl_3 +40μmol/L tBHQ group and 0.5 mmol/L LaCl_3 +40μmol/L tBHQ group increased significantly(P<0.05),except the expression level of SOD_2 protein in 0.25 mmol/L LaCl_3 +40μmol/L tBHQ group.Conclusion LaCl_3 can cause increase of ROS level,decrease of Nrf2,GST and SOD_2 protein expression,then lead to oxidative damage in neurons.tBHQ can antagonize the increase of ROS level,and the decrease of Nrf2,GST,SOD_2 protein expression,which may play a protective effect on the neurons.
Objective To explore antagonistic effects of tert-butyl hydroquinone(tBHQ)on oxidative damage of primary cerebral cortical neurons in rats induced by lanthanum chloride(LaCl_3 ).Methods The newborn Wistar rats within 24 hours were collected to performed primary neurons culture.Immunofluorescence(NSE)was used to identify the cultured neurons.Neurons were exposed to 0(control),0.125,0.25 and 0.5 mmol/L LaCl_3 in DMEM medium respectively,and also tBHQ(40μmol/L)+0.125mmol/L LaCl_3 ,tBHQ(40μmol/L)+0.25 mmol/L LaCl_3 and tBHQ(40μmol/L)+0.5 mmol/L LaCl_3 respectively for 24 hours respectively.Diacetyl dichlorofluorescein(DCFH-DA)was used to detect ROS levels of neurons.Western blot was used to measure Nrf2,GST and SOD_2 protein expression levels of neurons.Results Compared with the control group,ROS levels of primary neurons in 0.125,0.25 and 0.5 mmol/L LaCl_3 groups increased significantly(P<0.05),with a dose dependent manner.Compared with the related LaCl_3 exposure groups,ROS levels of primary neurons in 0.25 mmol/L LaCl_3 +40μmol/L tBHQ group and 0.5 mmol/L LaCl_3 +40μmol/L tBHQ group decreased significantly(P<0.05).Compared with the control group,Nrf2,GST and SOD_2 protein expression levels in 0.25 and 0.5 mmol/L LaCl_3 groups decreased significantly(P<0.05),except 0.125 mmol/L LaCl_3 group,and Nrf2,GST and SOD_2 protein expression levels gradually decreased with the increase of LaCl_3 exposure doses.Compared with the related LaCl_3 exposure groups,Nrf2,GST and SOD_2 protein expression levels of primary neurons in 0.125mmol/L LaCl_3 +40μmol/L tBHQ group,0.25 mmol/L LaCl_3 +40μmol/L tBHQ group and 0.5 mmol/L LaCl_3 +40μmol/L tBHQ group increased significantly(P<0.05),except the expression level of SOD_2 protein in 0.25 mmol/L LaCl_3 +40μmol/L tBHQ group.Conclusion LaCl_3 can cause increase of ROS level,decrease of Nrf2,GST and SOD_2 protein expression,then lead to oxidative damage in neurons.tBHQ can antagonize the increase of ROS level,and the decrease of Nrf2,GST,SOD_2 protein expression,which may play a protective effect on the neurons.
引文
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[24]Saykally JN,Rachmany L,Hatic H,et al.The nuclear factor erythroid2-like 2 activator,tert-butylhydroquinone,improves cognitive performance in mice after mild traumatic brain injury[J].Neuroscience,2012,223:305-314.
[25]Shih AY,Li P,Murphy TH.A small-molecule-inducible Nrf2-mediated antioxidant response provides effective prophylaxis against cerebral ischemia in vivo[J].J Neurosci,2005,25:10321-10335.
[2]Wang J,Zhou G.,Chen C,et al.Acute toxicity andbiodistribution of different sized titanium dioxide particles in mice after oral administration[J].Toxicol Lett,2007,168:176-185.
[3]徐厚恩.中国资源,环境与健康研究进展[M].北京大学医学出版社,2004:43-64.
[4]陈祖义,刘玉,王元兴.稀土元素铈(141Ce)在小鼠体内的分布与蓄积动态[J].南京农业大学学报,2000,23(3):101-103.
[5]刘玉,陈祖义,王元兴.钕在小鼠体内的分布与蓄积及对孕酮分泌的影响[J].中国稀土学报,2001,19(5):447-449.
[6]彭瑞玲,潘小川,解清.稀土矿区婴幼儿与其母亲头发中稀土含量的研究[J].中华预防医学杂志,2003,37(1):20-22.
[7]周宁,卢国,陈冠英,等.动物毛发与内脏中稀土元素含量的相关性研究[J].北京医科大学学报,1994,26(2):144-147.
[8]彭瑞铃,潘小川,解清,等.江西省婴幼儿稀土暴露与智能发育的相关性研究[J].中华预防医学杂志,2000,12(34增刊):55-57.
[9]范广勤,袁兆康,郑辉列,等.儿童稀土暴露的健康效应研究[J].卫生研究,2004,33(1):23-28.
[10]Feng LX,Xiao HQ,He X,et al.Neurotoxicological consequence of long-term exposure to lanthanum[J].Toxicol Lett,2006,165:112-120.
[11]Feng LX,Xiao HQ,He X,et al.Long-term effects of lanthanum intake on the neurobehavioral development of the rat[J].Neurotoxicol Teratol,2006,28:119-124.
[12]Yang J,Liu Q,Zhang LF,et al.Lanthanum chloride impairs memory,decreases pCaMK IV,p MAPK and p CREB expression of hippocampus in rats[J].Toxicol Lett,2009,190:208-214.
[13]杨敬华,刘秋芳,巫生文,等.生长发育期氯化镧暴露对大鼠神经元和神经突触超微结构的影响[J].环境与健康杂志,2010,27(8):659-661.
[14]陈祖义,朱旭东.稀土元素的骨蓄积性、毒性及其对人群健康的潜在危害[J].生态与农村环境学报,2008,24(1):88-91.
[15]曹睿,周青.稀土细胞毒理效应研究进展[J].中国生态农业学报,2007,15(4):180-184.
[16]陈祖义.稀土的hormesis效应及其农业应用对农业生态环境的潜在影响[J].农村生态环境,2004,20(4):1-5.
[17]Wu J,Yang JH,Liu QF,et al.Lanthanum induced primary neuronal apoptosis through mitochondrial dysfunction modulated by Ca2+and Bcl-2 family[J].Biol Trace Elem Res,2013,152:125-134.
[18]吴洁,刘秋芳,杨敬华,等.镧对原代神经元存活率和细胞内活性氧含量的影响[J].毒理学杂志,2010,24(6):427-427.
[19]Poli G,Leonarduzzi G,Biasi F,et al.Oxidative stress and cell signalling[J].Curr Med Chem,2004,11:1163-1182.
[20]Guyen T,Nioi P,Pickett CB.The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress[J].J Biol Chem,2009,284:13291-13295.
[21]Sburn WO,Kensler TW.Nrf2 signaling:an adaptive response pathway for protection against environmental toxic insults[J].Mutat Res,2008,659:31-39.
[22]Motohashi H,Yamamoto M.Nrf2-Keap1 defines a physiologically important stress response mechanism[J].Trends Mol Med,2004,10:549-557.
[23]Zhang LJ,Yang JH,Jin CH,et al.The effect of nuclear factor erythroid 2-related factor/antioxidant response element signaling pathway in the lanthanum chloride-induced impairment of learning and memory in rats[J].J Neurochem,2017,140:463-475.
[24]Saykally JN,Rachmany L,Hatic H,et al.The nuclear factor erythroid2-like 2 activator,tert-butylhydroquinone,improves cognitive performance in mice after mild traumatic brain injury[J].Neuroscience,2012,223:305-314.
[25]Shih AY,Li P,Murphy TH.A small-molecule-inducible Nrf2-mediated antioxidant response provides effective prophylaxis against cerebral ischemia in vivo[J].J Neurosci,2005,25:10321-10335.