摘要
适量氟是维持机体正常生理活动的基础,但氟是一种蓄积性原生质毒物,机体长期过量摄入氟可引起氟骨症、氟斑牙等骨相器官的损害以及血液、神经系统等非骨相器官的损害。神经组织对氟的敏感性很高,海马作为中枢神经系统中氟中毒的靶部位之一,其神经细胞易受氟的影响而发生可塑性变化。近年来,通过对氟神经毒性作用机制的研究,人们主要提出了G蛋白信号转导理论与氧化应激理论。
目前普遍认为,神经毒物引起的神经系统损伤与G蛋白信号转导通路中Ca2+稳态失调有密切关系。其机制可能与神经细胞内对氧化应激敏感且受钙离子调控的核转录因子(nuclear factor kappa-B, NF-κB)表达等有关。因此,本实验通过复制饮水型慢性氟中毒动物模型,以观察和检测氟中毒引起氟斑牙、血氟水平异常及海马神经细胞形态结构改变为基础,首次在氟的神经毒性研究中将钙离子信号通路与氧化应激理论结合,检测氟中毒对大鼠海马突触体内Ca2+浓度以及钙离子信号通路中CaMKⅡ、c-fos、NF-κB、Bcl-2及Bax蛋白表达的影响。
研究方法:152只初断乳雄性SD大鼠随机分为四组,即对照组(饮用自来水,水氟含量低于0.5mg/L),低氟组、中氟组、高氟组(饮水含氟化钠分别为15mg/L、30mg/L、60mg/L)。各组以配制的溶液作为饮水唯一来源,自由摄食、饮水,饲养时间为18个月。染氟初期(3月龄)对大鼠进行氟斑牙和血氟检测,染氟中期(9月龄)和染氟结束(18月龄)分两批断头处死大鼠,HE染色检测海马CA3区病理学改变,荧光指示剂Fura-2/AM检测慢性氟中毒大鼠海马突触体内Ca2+浓度的变化,免疫组化法检测海马CA3区CaMKⅡα、c-fos、NF-κBρ65、Bcl-2及Bax蛋白表达的改变。
1、过量氟摄入会导致大鼠氟斑牙发生,随染氟浓度升高,大鼠氟斑牙症状加剧,切齿明显变形、缺损并出现白垩状横纹;各染氟组血氟浓度与对照组相比显著或极显著升高(P<0.05或P<0.01)。表明动物氟中毒模型复制成功。
2、HE染色结果显示,慢性氟中毒可损伤大鼠神经细胞形态结构。与对照组相比,染氟组大鼠海马神经细胞分布稀疏,细胞轮廓消失,并出现空泡。且这种变化随染氟时间和染氟浓度的递增而更为显著。
3、突触体Ca2+浓度检测结果显示,随大鼠月龄增长,对照组突触体Ca2+浓度有升高的趋势,而染氟处理加剧了Ca2+的超载。染氟中期:与对照组相比,中氟组、高氟组突触体Ca2+浓度显著或极显著升高(P<0.05或P<0.01);染氟结束:与对照组相比,中氟组、高氟组突触体Ca2+浓度极显著升高(P<0.01)。
4、IHC结果显示,随大鼠月龄增长,对照组及各染氟组CaMKⅡα、c-fos、NF-κBρ65蛋白表达均有不同水平的上升,且染氟时间和染氟剂量的递增使这种变化更为显著。染氟中期:与对照组相比,中氟组、高氟组CaMKⅡα表达显著升高(P<0.05),c-fos表达显著或极显著升高(P<0.05或P<0.01),NF-κBρ65表达虽无显著性差异,但随染氟剂量升高其表达有上升的趋势;染氟结束:与对照组相比,中氟组、高氟组CaMKⅡαc-fos表达极显著升高(P<0.01),中氟组NF-κBρ65表达显著升高(P<0.05)。
5、IHC结果显示,随大鼠月龄增长,对照组及各染氟组Bcl-2和Bax蛋白表达水平均有不同水平的下降,且染氟时间和染氟剂量的递增使这种变化更为显著。染氟中期:与对照组相比,中氟组、高氟组Bcl-2表达显著或极显著下降(P<0.05或P<0.01),Bax表达极显著下降(P<0.01);染氟结束:与对照组相比,中氟组、高氟组Bcl-2表达显著或极显著下降(P<0.05或P<0.01),低氟组、中氟组、高氟组Bax表达显著或极显著下降(P<0.05,P<0.01,P<0.01)。同时,染氟中期:Bax与Bcl-2比值(Bax/Bcl-2)随染氟时间和染氟浓度的递增有升高的趋势;染氟结束:与对照组相比,中氟组、高氟组Bax/Bcl-2比值显著上升(P<0.05),提示神经细胞处于凋亡状态。
综合上述,慢性氟暴露对中枢神经系统有毒性作用。氟可导致海马神经细胞形态结构发生改变,引起海马突触体Ca2+超载,继而导致钙离子信号通路中CaMKⅡα、c-fos、NF-κBρ65、Bcl-2及Bax的蛋白表达异常,最终诱导神经细胞趋于凋亡。衰老过程中,神经细胞的退行性改变也可导致大鼠神经细胞凋亡,而染氟处理加剧了这种变化。本研究进一步阐明了氟神经毒性对钙离子信号通路影响的分子机理,为深入探讨慢性氟中毒对中枢神经系统毒性作用的分子生物学机制提供了一定的理论基础。此外,NF-κB的表达与染氟时间、浓度有敏感的量效关系,可能是氟神经毒性相关靶分子,这为开展氟神经毒性的生物标志物研究提供一定的理论依据。
Moderate fluoride is the foundation of normal physiological activities. But the fluoride is a long-term accumulated poison, and excess intake of fluorine can cause skeletal fluorosis, dental fluorosis so far as to blood and nervous system damage. Nerve tissues are sensitive to fluorine particularly. Hippocampus as a target site of fluorosis in central nervous systems, which synaptic plasticity of nerve cells is vulnerable by affecting fluorine. In recent years, through the study on neurotoxic mechanism of fluorine, people put forward two theories that the theory of G protein signal transduction and the theory of oxidative stress.
Generally thought, there is a close relation between the nervous system damage caused by nerve poison and Ca2+ steady-state disorder in G protein signal transduction pathways. The mechanism may relate to Nuclear factor-κB (NF-κB) which is sensitive in oxidative stress and regulated by Ca2+. Thus, we chose rat model of chronic fluorosis to explore the effects of different doses of fluoride on dental fluorosis, blood F and nerve cell structure firstly. Then combining calcium signaling pathways theory with oxidative stress theory for the first time, we detected calcium ion concentration ([Ca2+]) and the expression level of CaMKⅡ, c-fos, NF-κB, Bcl-2 and Bax.
152, just weaning male Sprague-Dawley rats were randomly divided into four groups and given 15,30,60 mg/L NaF solution and distilled water respectively for 9 and 18 months. Rats were fed standard diet throughout the experiment and water was given ad libitum. The degree of dental fluorosis and blood F were tested when three months; animals were killed by cervical when exposure was stopped, then evaluated pathological change in hippocampus with HE staining, [Ca2+] in synaptosomes was measured by double wavelength fluorescence spectrophotometer, and expression level of CaMKⅡα、c-fos、NF-κBρ65、Bcl-2、Bax in hippocampus CA3 region with immunohistochemistry (IHC) method.
1. As the concentration of fluoride increased, blood F increased in experimental groups and had differences or significant differences (P<0.05, P<0.01) compared with the control group, and the level and symptom of dental fluorosis in rats had increased. The teeth of high fluoride group rats became deformed and defected to some degree. Both indices also show that the fluorosis model worked in the research.
2. Chronic fluorosis can impair rat's brain nervous cells. HE staining showed that, the distribution of rat's hippocampus pyramidal cells were sparse, cell contour disappeared and vacuole in cells.
3. The test of [Ca2+] in synaptosomes showed that, synaptosomes [Ca2+] increased in the experimental groups with the fluoride concentration increased. Compared with the control group, moderate group had significant difference (P<0.01) and high-fluoride group had difference (P<0.05) in middle-term; moderate group and high-fluoride group had significant difference (P<0.01) in the end of experiment.
4. IHC staning showed that, the expression level of protein CaMKⅡα、c-fos and NF-κBρ65 increased with the growth of fluoride concentration. Compared with control group, CaMKⅡαmoderate group and high-fluoride group had difference (P<0.05), c-fos moderate group and high-fluoride group had difference or statistical difference (P<0.05 or P<0.01), NF-κBρ65 had no significant difference in middle-term; CaMKⅡαand c-fos moderate group and high-fluoride group had statistical difference (P<0.01), NF-κBρ65 moderate group had difference (P<0.05) in the end of experiment.
5. IHC staning also showed that, the expression level of protein Bcl-2 and Bax decreased with the growth of fluoride concentration. Compared with control group, Bcl-2 moderate group and high-fluoride group had difference or significant difference (P<0.05 or P<0.01), Bax moderate group and high-fluoride group had significant difference (P<0.01) in middle-term; Bcl-2 moderate group had difference (P<0.05), high-fluoride group had significant difference (P<0.01), Bax low-fluoride group had difference (P<0.05), moderate group and high-fluoride group had significant difference (P<0.01) in the end of experiment. And Bax/Bcl-2 moderate group and high-fluoride group had increased (P<0.05) in the end of experiment, compared with control group.
Above all, chronic fluoride exposure had toxic effect on the central nervous system. Chronic fluorosis could injure the nerve cells in the hippocampus CA3 region to some degree. Our results show that the hippocampus synaptosomes [Ca+] were overloaded and the expression level of CaMKⅡα、c-fos、NF-κBρ65、Bcl-2 and Bax were abnormally in experimental groups. So chronic fluorosis could induction nerve cells tend to apoptosis eventually. Degenerative change also can cause nerve cell apoptosis, and fluorine aggravated this change. The study on Ca2+ channel signal may be the molecular basis of central nervous system damage by fluorosis. And NF-κB may be one of the related molecular target factors in central nervous system damage by fluorosis. So we can make some deeper research in early-animal-development and it can offer us a theoretical basis in research of NF-κB as an environmental neurotoxic substance biomarker.
引文
[1]Mandinic Z, Curcic M, Antonijevic B, et al. Relationship between fluoride intake in Serbian children living in two areas with different natural levels of fluorides and occurrence of dental fluorosis [J]. Food and Chemical Toxicology,2009,47(6): 1080-1084.
[2]Xiong XZ, Liu JL, He WH, et al. Dose-effect relationship between drinking water fluoride levels and damage to liver and kidney functions in children [J]. Environ-mental Research,2007,103(1):112-116.
[3]Niu RY, Sun ZL, Cheng ZT, et al. Decreased learning ability and low hippocampus glutamate in offspring rats exposed to fluoride and lead [J]. Environmental Toxicology and Pharmacology,2009,28(2):254-258.
[4]Li HR, Liu QB, Wang WY, et al. Fluoride in drinking water, brick tea infusion and human urine in two counties in Inner Mongolia, China [J]. Journal of Hazardous Materials,2009,167(1-3):892-895.
[5]Ando M, Tadano M, Yamamoto S, et al. Health effects of fluoride pollution caused by coal burning [J]. The Science of the Total Environment,2001,271(1-3):107-116.
[6]Cao J, Zhao Y, Liu JW, et al. Prevention and control of brick-tea type fluorosis-a 3-year observation in Dangxiong, Tibet [J]. Ecotoxicology and Environmental Safety,2003,56(2):222-227.
[7]Patra RC, Dwivedi SK, Bhardwaj B, et al. Industrial fluorosis in cattle and buffalo around Udaipur, India [J]. The Science of the Total Environment,2000,253(1-3): 145-150.
[8]张璟,祝文静,章子贵.慢性氟中毒与脑损伤关系研究进展[J].中国公共卫生,2010,26(2):0251-0252.
[9]Mehmet NT, Banu KK, Cagatay A, et al. Osteosclerosis due to endemic fluorosis [J]. Science of the Total Environment,2007,373(1):43-48.
[10]Fordyce FM, Vrana K, Zhovinsky E, et al. A health risk assessment for fluoride in Central Europe [J]. Environ Geochem Health,2007,29(2):83-102.
[11]王环增,沈尔礼,候培森,等.中国地方性氟中毒防治工作进展副[M].第20届国际氟研究协会学术讨论会论文集,中国北京,1994:23-25.
[12]王夔主编.生命科学中的微量元素.第二版.中国计量出版社,1996:25-28.
[13]Dryzek H. Electronic version of the encyclopaedia of occupational health and safety as a source of definitions [J]. Journal of Safety Research,2002,33(2):155-163.
[14]詹承烈.关于氟病的发病机理问题[J].中国职业医学,1990,17(4):244-246.
[15]Whitford GM, Williams JL. Fluoride absorption:Independence from plasma fluoride levels [J]. Fluoride,1987,20(3):46-49.
[16]Kumar H, Boban M, Tiwari M. Skeletal fluorosis causing high cervical myelo-pathy [J]. Journal of Clinical Neuroscience,2009,16(6):828-830.
[17]李广生.从“钙矛盾”看氟骨症的发生机制[C].全国氟砷学术会议论文集,哈尔滨:中国地方病学杂志编辑部.
[18]任立群,李广生,孙波.中长周期慢性氟中毒对大鼠骨转换的影响及机理研究[J].中国地方病学杂志,1998,17(2):13-16.
[19]吴天秀,廖进民,陈艳,等.氟化物对幼年大鼠骨胶原的影响[J].现代医院,2009,12(9):11-13.
[20]康龙丽,郭雄,许鹏,等.“硼复方”对过量氟实验大鼠血清脂质过氧化和RNA合成的影响[J].西安交通大学学报,2003,24(6):599-601.
[21]李省,陈悦,许启泰,等.自由基与氟中毒对RNA代谢影响[J].中国公共卫生,2007,23(1):113-115.
[22]桂传枝,王长松,于燕妮,等.氟对软骨细胞与成骨细胞凋亡及H2O2、SOD、NO的影响[J].中国地方病学杂志,2004,23(2):108-112.
[23]Whyte MP, Essmyer K, Gannon FH, et al. Skeletal fluorosis and instant tea [J]. The American Journal of Medicine,2005,118(1):78-82.
[24]Kobayashi CA, Leite AL, Silva TL, et al. Proteomic analysis of kidney in rats chronically exposed to fluoride [J]. Chemico-Biological Interactions,2009,180(2): 305-311.
[25]Wang YY, Zhao BL, Li XJ, et al. Spintrapping technique studies on active oxygen radicals from human polymorphonuclear leukocytes during fluoride-stimulated respiratory burst [J]. Fluoride,1997,30(1):5-15.
[26]陈百炼,李恩东,戴国钧.慢性氟中毒患者红细胞形态损伤的电镜观察[J].中日友好医院学报,2005,19(4):224-227.
[27]张婕,冀芳.氟砷联合中毒患者无机元素和胶原代谢状况观察[J].地方病通报,2005,20(1):21-23.
[28]张晨,刘继文,凌冰,等.氟砷染毒对大鼠子代脂质过氧化及微量元素的影响[J].新疆医科大学学报,2004,27(2):116-118.
[29]孙玉霞.氟对神经系统的毒性[J].中国地方病学杂志,2008,23(3):184-185.
[30]孙增荣,刘风贞,杨海贤,等.饮高氟水小鼠脑海马回组织超微结构观察[J].中国地方病学杂志,2000,19(5):333-334.
[31]Zhang ZG, Xu XL, Shen XY, et al. Effect of fluoride exposure on synaptic structure of brain areas related to learning-memory in mice [J]. Fluoride,2008, (41):139-143.
[32]张本忠,吴德生,无机氟对大小鼠胚胎中脑细胞增殖和分化的影响[J].中国地方病防治杂志,1998,13(3):129-131.
[33]Vani ML, Reddy KP. Effeets of fluoride accumulation on some enzymes of brain and gastrocnemius musele of mice [J]. Fluoride,2000,33(2):17-26
[34]Hanen B, Francoise C, Tahia B, et al. Oxidative stress induced by fluoride in adult mice and their suckling pups [J]. Experimental and Toxicologic Pathology,2007, (58):339-349.
[35]许晓路,章子贵,申秀英.氟中毒对小鼠学习记忆行为及脑内SOD活性和MDA含量的影响[J].中国公共卫生,2001,17(1):8-10.
[36]Ekambaram P, Paul V. Calcium orevernting locomotor behavioral and dental toxicities of fluoride by decreasing serum fluoride level in rats [J]. Fluoride,2001, 9(4):141-143
[37]章子贵,许晓路,申秀英.硒对氟致小鼠学习记忆损伤的改善作用[J].卫生研究,2001,30(3):144-146.
[38]Liu YJ, Gao Q, Wu CX, et al. Alterations of nAChRs and ERK1/2 in the brains of rats with chronic fluorosis and their connections with the decreased capacity of learning and memory [J]. Toxicology Letters,2010,192(3):324-329.
[39]张璟,祝文静,章子贵.孕鼠氟暴露对仔鼠学习记忆能力影响[J].中国公共卫生,2009,25(11):1347-1348.
[40]Gui CZ, Ran LY, Li J, et al. Changes of learning and memory ability and brain nicotinic receptors of rat offspring with coal burning fluorosis [J]. Neuro- toxicology and Teratology, online first.
[41]李赫.氟对胎儿儿童生长发育和智离的影响[J].中国地方病防治杂志,1992,7(7):155.
[42]王正辉,王三祥,张向东,等.长期氟暴露儿童生长发育的调查研究[J].中国地方病防治杂志,2006,21(4):239-241.
[43]Xing Q, Liang Y, Chen L, et al. Effect of fluoride in drinking water [J]. Fluoride, 2003,36(2):84-94.
[44]Lu Y, Sun ZR, Wu LN, et al. Effect of hight-fluoride water on intelligence in children [J]. Fluoride,2000,33(2):274-278.
[45]Hkano S, Ando M. Apoptotic cell death following exposure to fluoride in rat alveolar macrophages [J]. Archives Toxicol,1996,70(3-4):249-251.
[46]Schwarze PE, Johnsen NM, Samuelsen JT, et al. The use of isolated lung cells in vitro pulmonary toxicology:study of DNA damage, apoptosis and alteration of gene expression [J]. Central European Journal Public Health,1996,4(1):6-10.
[47]Cicek E, Aydin G, Akdogan M, Okutan H. Effects ofchronic ingestion of sodium fluoride on myocardium in a second generation of rats [J]. Human and Experimental Toxicol,2005,24(2):79-87.
[48]陶新,许梓荣,汪以真.氟中毒对动物健康影响的研究进展[J].中国畜牧杂志,2006,42(3):61-63.
[49]Shashi A. Histopathological effects of sodium fluoride on the duodenum of rabbits [J]. Fluoride,2002,35(1):28-37.
[50]谢永平,葛相金,姜玉辛,等.高氟对人体胰岛细胞功能影响的研究[J].中国地方病学杂志,2000,19(2):84-86.
[51]郑继伟,莫志亚.地方性氟中毒病区与非病区老年性白内障与超氧化物歧化酶[J].中国地方病预防杂志,1999,14(4):228-229.
[52]郭晓英,陈爱莉,孙贵范.氟对大鼠肝脏氧化应激及超微结构的影响[J].中国医科大学学报,2005,34(4):321-322.
[53]张桂珍,高申,郑德明,等.过量氟对大鼠脑肝肾细胞周期与细胞凋亡的影响[J].中国地方病学杂志,2001,20(5):344-346.
[54]边晓燕,张守臣,王志成,等.实验性氟中毒大鼠肝、肾组织的超微结构观察[J].电子显微学报,2000,19(3):227-228.
[55]申秀英,许晓路,章子贵,等.碘硒氟联用对小鼠肝肾脂褐素含量的影响[J].浙江师范大学学报(自然科学版),2003,26(1):51-52.
[56]Dvorakova-Hortova K, Sandera M, Jursova M, et al. The influence of fluorides on mouse sperm capacitation [J]. Animal Reproduction Science,2008,108(1-2):157-170.
[57]辛伟红,朱岱,张爱君,等.氟中毒大鼠听觉脑干诱发反应特点[J].中国地方病杂志,2008,23(4):248-250.
[58]张爱君,吴占河.细胞凋亡与氟中毒[J].中国地方病防治杂志,2003,18(1):31-34.
[59]王俊玲.氟化钠导致小鼠神经细胞凋亡的研究[J].毒理学杂志,2007,21(5):420-421.
[60]Susa M. Heterotrimeric G protein as fluoride targets in bone [J]. International Journal of Medical,1999,3 (2):115-126
[61]Refsnes M, Schwarze PE, Holme JA, et al. Fluoride-induced apoptosis in human epithelial lung cells (A 549 cells):role of different G protein-linked signal systems [J]. Human and Experiment Toxicology,2003,22 (3):111-123.
[62]刘俊玲,王爱国.氟化物诱导细胞凋亡机制的研究进展[J].国外医学地理分册,2004,25(2):49-77.
[63]Park IC, Park MJ, Rhee CH, et al. Protein kinase C activation by PMA rapidly induces apoptosis through caspase-3/CPP32 and serine protease(s) in a gastric cancer cell line [J]. International Journal Oncology,2001,18(5):1077-1083.
[64]Caverzasio J, Palmer G, Suzuki A, et al. Mechanism of the mitogenic effect of fluoride on osteoblast-like cells:evidences for a G protein-dependent tyrosine phosphorylation process [J]. Journal of Bone Mineral Research,1997,12(12): 1975-1983.
[65]Thrane EV, Refsnes M, Thoresen GH, et al. Fluoride-induced apoptosis in epithelial lung cells involves activation of MAP kinases p38 and possibly JNK [J]. Toxicol Science,2001,61(1):83-91.
[66]Ranjan R, Swarup D, Patra R C, et al. Oxidative stress indices in erythrocutes, liver, and kidneys of fluoride-exposed rabbits [J]. Fluoride,2009,42(2):88-93.
[67]Rzeuskl R, Chlubek D, Machoy Z, et al. Interactions between fluoride and biological free radical reaction [J]. Fluoride,1998,31(1):43-45.
[68]Shiyaraiashankara YM, Shiyaraiashankara AR, Gopalakrishna BP, et al. His-tological changes in the brain of young fluoride-intoxicated rats [J]. Fluoride,2002, 35(1):12-21.
[69]Zhang M, Wang AG, He WH, et al. Effects of fluoride on the expression of NCAM, oxidative stress, and apoptosis in primary cultured hippocampal neurons [J]. Toxicology,2007,236 (3):208-216.
[70]Anuradha CD, Kanno S, Hirano S. Oxidative damage to mitochondria is a pre-liminary step to caspase-3 activation in fluoride-induced apoptosis in HL60 cells [J]. Free Radical Biology and Medicine,2001,31(3):367-373.
[71]呼亚伟,催炳元,张津旗,等.氟中毒大鼠肝肾细胞凋亡与p53的关系[J].中国地方病防治杂志,2002,17(3):141-142.
[72]陈军,陈学敏,杨克敌,等.氟对大鼠脑细胞DNA损伤及诱导凋亡的作用[J].中华预防医学杂志,2002,36(4):222-224.
[73]Wang AG, Chu QL, He WH, et al. Effects on protein and mRNA expression level of p53 induced by fluoride in human embronichepatocytes [J]. Toxicol Letter, 2005,158(2):158-163.
[74]Kaufmann JA, Biekford PC, Taglialatela G. Free radical-dependent changes in constitutive nuclear factor kappa bin the aged hippocampus [J]. Neuroreport,2002, 3(5):1917-1928.
[75]孙元明,杨福军,李雨民.细胞凋亡信息分子Fas、FasL和NF-κB在氟化物作用后破骨样细胞凋亡过程中的表达[J].中国医学科学学报,2002,24(5):491-494.
[76]吕晓红,吕晓丽,李广生.慢性氟中毒鼠神经元与细胞凋亡相关基因的研究[J].中国地方病学杂志,2003,22(2):119-121.
[77]吴天秀,廖进民,陈艳,等.氟化物对幼年大鼠骨矿物质和血微量元素的影响极其相关性研究[J].广东微量元素科学,2009,16(9):13-19.
[78]刘国艳,张维东,顾金辉,等.氟对大鼠甲状腺结构和功能的影响[J].上海交通大学学报(农业科学版),2008,26(69):537-539.
[79]李广生.进一步深入研究慢性氟中毒的发病机制[J].中华预防医学杂志,2003,37(4):225-226.
[80]Thomas HL, Alexander H, Baltimore D. One nucleotide in a κB site can determine cofactor specificity for NF-κB dimmers [J]. Cell,2004,118(4):453-457.
[81]Zhang AH, Hao S, Bi J, et al. Effects of catalpol on mitochondrial function and working memory in mice after lipopolysaccharide-induced acute systemic inflammation [J]. Experimental and Toxicologic Pathology,2009,61(5):461-469.
[82]Murao H, Sakagami N, Iguchi T, et al. Sodium fluoride increases intracellular calcium in rat renal epithelial cell line NRK-52E [J]. Biological and Pharma-ceutical Bulletin,2000,23(5):581-593.
[83]张均田.老化、老年痴呆与钙代谢失衡及其治疗新途径[J].药学学报,1993,28(9):641-646.
[84]Fanny Longpre, Philippe G, Yves C, et al. Protection by EGb 761 against P-amyloid-induced neurotoxicity:Involvement of NF-κB, SIRT1, and MAPKs pathways and inhibition of amyloid fibril formation [J]. Free Radical Biology and Medicine,2006,41(12):1781-1793.
[85]Xiang QY, Zhou MH, Wu M, et al. Relationships between daily total fluoride intake and dental fluorosis and dental caries [J]. Journal of Nanjing Medical University,2009,23(1):33-39.
[86]Yan Q, Zhang Y, Li W, et al. Mieromolar fluoride alters ameloblast lineage cells in vitro [J]. Journal Dental Research,2007,86(4):336-340.
[87]黄安斌,陈荣安.微量法检测指血和血清氟的分析评价[J].卫生研究,2002,31(1):65-66.
[88]万桂敏,莫志亚.地方性氟中毒患者多项检验指标的测定及分析[J].中国地方病学杂志,2001,20(2):137-139.
[89]Tamer MN, Koroglu BK, Cagatay A, et al. Osteosclerosis due to endemic fluorosis [J]. Science of the Total Environment,2007,373 (1):43-48.
[90]万桂敏,王守智,徐春蓓,等.血清中无机氟测定标准方法的研究[J].中国地方病防止杂志,2000,15(5):266-269.
[91]肖邦良,董奇男,李守祺,等.大鼠摄入铝、氟极其不同比例混合物后血和骨中福含量分析[J].华西医大学报,1992,23(2):185-189.
[92]Kukleva MP, Isheva AV, Kondeva VK, et al. Prevalence of dental fluorosis among 4-to 14-year-old children from the town of Dimitrovgrad (Bulgaria) [J]. Folia Medica (Plovdiv),2007,49(1-2):25-31.
[93]孙仲楠,张抒,李亮.直接贴面法治疗氟斑牙的临床疗效观察[J].中周地方病学杂志,2007,26(4):462-464.
[94]孙宏晨,刘明,欧阳喈,等.过量氟对大鼠切牙成釉细胞增生和凋亡的影响[J].中华口腔医学杂志,2003,38(3):217-219.
[95]Browne D, Whehon H, O'Mullane D. Fluoride metabolism and fluorosis [J]. Journal of Dentisity,2005,33(3):177-186.
[96]陶荣,宁钢民,杨勇,等.培养的大鼠海马神经元胞内Ca2+和NO双标记方法研究[J].生物化学与生物物理进展,2005,32(8):788-793.
[97]赵岩, 刑伟.铝对学习记忆及大鼠海马 Ca2+-CaM-CaMKⅡ途径的影响 [J]. 生命的化学,2007,27(6):530-533.
[98]Zhao L, Zhao Q, Lu R, et al. Effects of tyroserleutide on gene expression of calmodulin and PI3K in hepatocellular carcinoma [J]. Cell Biochem,2008,103(2): 471-478.
[99]Shen XC, Valencia CA and Gao WY, et al., Ca2+/calmodulin-binding proteins from the C. elegans proteome [J]. Cell Calcium,2008,43(5):444-456.
[100]刘振伟,王福生,要航,等.用Fura-2/AM测定缺氧时细胞内游离钙离子浓度的变化[J].细胞生物学杂志,1994,16(3):141.
[101]Li S, Zhao ZY, Zhou XL, et al. The effect of lead on intracellular Ca2+ in mouse lymphocytes [J]. Toxicology in Vitro,2008,22(8):1815-1819.
[102]Su JJ, Osoegawa M, Matsuoka T, et al. Upregulation of vascular growth factors in multiple sclerosis:correlation with MRI findings [J]. Neurological Sciences, 2006,243(1):21-30.
[103]陈燕启,刘德红,杨光田.大鼠急性全脑缺血再灌注时NF-κB表达及意义[J].医学临床研究,2004,21(7):772.
[104]章子贵,许晓路,申秀英,等.氟中毒对小鼠学习记忆相关脑区突触结构的影响[J].卫生研究,1999,28(4):210-212.
[105]Ehrich M, Wu HL, Were SR, et al. Calcium signaling in neuronal cells exposed to the munitions compound cyclotrimethylenetrinitramine (RDX) [J]. International Journal of Toxicology,2009, (28):425-435.
[106]Concepcion NR, Mireya AZ, Marisela MA, et al. Nitric oxide synthase immuno-localization and expression in the rat hippocampus after sub-acute lead acetate exposure in rats [J]. Experimental and Toxicologic Pathology, online first.
[107]Harukuni I, Bhardwaj A. Mechanisms of brain injury after global cerebral ischemia [J]. Neurology Clinic,2006,24(1):1-21.
[108]章子贵,徐晓虹,杜红燕,等.衰老性记忆障碍与脑突触体内游离[Ca2+]i的相关性[J].生理学报,2000,52(1):85-88.
[109]Zhang J, Zhu WJ, Xu XH, et al. Effect of fluoride on calcium ion concentration and expression of nuclear transcription factor kappa-B ρ65 in rat hippocampus [J]. Experimental and Toxicologic Pathology, online first.
[110]Sobczak M, Blazejczyk, Piszczek G, et al. Calciumbinding calmyrin forms stable covalent dimers in vitro, but in vivo is found in monomeric form [J]. Acta Biochimica Polonica,2005,52 (2):469-476.
[111]Babcock AM, Standing D, Bullshields K. In vivoinhibition of hippocampal Ca2+ calmodulin-dependent protein kinase Ⅱ by RNA interference [J]. Molecular Therapy,2005,11(6):899-905.
[112]Liu Y, Templeton D M. Cadmium activates CaMK-II and initiates CaMK-II-dependentapoptosis inmesangial cells [J]. Federation of European Biochemical Societies Letters,2007,581(7):1481-1486.
[113]Gubellini P, Ben-Ari Y, Gaiarsa JL. Endogenous neurotrophins are required for the induction of GABAergic long-term potentiation in the neonatal rat hippocampus [J]. The Journal of Neuroscience,2005,25(24):5796-5802.
[114]倪宏,姜玉武,陶陆阳,等.新生期反复惊厥对认知和海马CaMKII表达的远期影响及干预研究[J].生物化学与生物物理进展,2008,35(1):97-104.
[115]Morgan JI, Curran T. Stimulus transcription coupling in the nervous system Involvement of the inducible proto-oncogenes fos and jun [J]. Annual Review of Neuroscience,1991,14:421-451.
[116]徐康.自由基对转录因子NF-κB活性的调节[J].医学研究生报,2004,17(7):654-656.
[117]Memet S. NF-kappaB functions in the nervous system:from development to disease [J]. Biochemical Pharmacology,2006,72(9):1180-1195.
[118]Zhang M, Wang AG, Xia T, et al. Effects of fluoride on DNA damage, S-phase cell-cycle arrest and the expression of NF-κB in primary cultured rat hippocampal neurons [J]. Toxicology Letters,2008,179(1):1-5.
[119]Gutierrez H, Hale VA, Dolcet X, et al. NF-kappaB signaling regulates the growth of neural processes in the developing PNS and CNS [J]. Development,2005,132 (7):1713-1726.
[120]Torricelli C, Fortino V, Capurro E, et al. Rottlerin inhibits the nuclear factor κB/cyclin-D1 cascade in MCF-7 breast cancer cells [J]. Life Sciences,2008,82 (11-12):638-643.
[121]Joel LP, Baltimore D. Two Pathways to NF-κB [J]. Molecular Cell,2002,10: 693-701.
[122]Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences [J]. Cell,1986,46(50):705-716.
[123]史艳晖,卢圣栋.转录因子NF-κB的研究现状及其应用前景[J].中国生物工程杂志,2007,27(4):104-110.
[124]Read MA, Whitley MZ, Williams AJ, et al. NF-kappa B and I kappa B alpha:an inducible regulatory system in endothelial activation [J]. J Exp Med,1994, 179(2):503-512.
[125]张劲松,王兴宇,单佑安,等.转录因子NF-κB的研究进展[J].科学通报,2002,47(5):323-329.
[126]Meffert MK, Chang JM, Wiltgen BJ, et al. NF-κB functions in synaptic signaling and behavior [J]. Nature Neuroscience,2003,6(10):1072-1078.
[127]Cruise L, Ho LK, Veitch K, et al. Kainate receptors activate NF-κB via MAP kinase in striatal neurons [J]. Neuroreport,2000,11 (2):395-398.
[128]张运阁,陶陆阳.NF-κB与神经系统损害的研究进展[J].法医学杂志,2008,24(6):453-456.
[129]Denk A, Wirth T, Baumann B. NF-kappaB transcription factors:critical re-gulators of hematopoiesis and neuronal survival [J]. Cytokine and Growth Factor Reviews,2000,11(4):303-320.
[130]Mattson MP. NF-kappaB in the survival and plasticity of neurons [J]. Neuro-chemical Research,2005,30(6-7):883-893.
[131]王长松,刘家骝,于燕妮,等.氟化物对细胞凋亡的影响及其分子机制[J].国外医学地理分册,2003,24(4):145-151.
[132]杨发明,董联玲,曹娟,等.红景天对血管性痴呆大鼠行为学及海马CA1区Bcl-2及Bax表达的影响[J].中国中医药信息杂志,2008,15(6):39-41.
[133]贾丽玲,曹定睿,张维智,等.瑞芬太尼预处理对大鼠脑缺血再灌注后神经细胞凋亡及Bcl-2、Bax表达的影响[J].中国药物与临床,2010,10(2):171-173.