【作者】 张舜；

【导师】 王爱国；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2014， 博士

【摘要】 第一部分葡萄糖利用减少和神经退行性改变参与氟暴露引起的大鼠认知损伤目的：氟是一种较活泼的微量元素,除了导致氟骨症和氟斑牙、引起骨代谢紊乱外,还能对其它非骨相器官产生严重的损害。近年来,氟对神经系统的发育毒性引起了人们的广泛关注,但其具体的作用机制还不十分清楚。本研究旨在探讨氟暴露所致发育神经毒性的潜在发生机制。方法：将40只清洁级成年Sprague-Dewley (SD)大鼠(雌雄各半)随机分成四组：对照组(自来水,氟含量低于1mg/L)和25mg/L、50mg/L、100mg/L三个氟化钠剂量组(氟化钠溶解于自来水中),自由饮水染毒10天后,雌、雄大鼠按1：1合笼,受孕期间继续按照上述剂量染毒至仔鼠断乳(出生后第21天),将所有仔鼠按性别和剂量分开,接受与亲代相同的染毒处理,到仔鼠出生后第8周末(出生后第56天)结束。采用Morris水迷宫检测子代大鼠的空间学习记忆能力；应用小动物磁共振成像(Magnetic resonance imaging,MRI)技术观察活体大脑形态学改变；小动物正电子发射断层显像／计算机断层扫描(Positron emission tomography/Computed tomography, PET/CT)活体检测脑葡萄糖利用情况；电镜下观察海马超微结构改变;Western blot方法检测脑功能相关基因葡萄糖转运蛋白1(glucose transporterl,GLUT1)、脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)以及胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)的蛋白表达水平。结果：与对照组相比,25mg/L、50mg/L和100mg/L氟暴露均可明显增加子代大鼠到达平台的潜伏期和路径,并显著降低大鼠在平台所在象限的游行时间(路径)和总时间(总路径)的比值,且各组内雌雄之间无性别差异；MRJ检测结果显示染氟大鼠侧脑室T2加权信号增强,提示染氟大鼠出现脑室扩张；PET/CT成像检测结果表明,50mg/L和100mg/L两个染氟组子代大鼠脑葡萄糖利用率明显降低；电镜下超微结构显示氟可导致海马CA1区大量空泡化形成、神经元显著变性并发生脱髓鞘改变；脑功能基因检测发现,25mg/L、50mg/L和100mg/L氟处理均可明显下调大鼠脑皮层和海马GLUT1和GFAP的表达水平,并显著上调皮层和海马BDNF的表达水平,上述三个蛋白在相同暴露剂量的雌雄大鼠间的表达水平相近。结论：发育期氟暴露引起的神经毒性与大脑葡萄糖利用减少和神经退行性改变密切相关。第二部分内质网应激和炎症反应在氟暴露所致大鼠睾丸损伤中的作用目的：长期过量氟暴露可对机体多种组织器官造成损伤。虽然氟对雄性生殖系统的损伤作用已被确认并备受人们关注,但氟导致生殖毒性的具体作用机制还未完全阐明。本研究通过模拟人群氟暴露方式建立动物氟暴露模型,旨在探讨内质网应激和炎症反应在氟暴露致子代大鼠睾丸损伤中的作用。方法：将40只清洁级成年Sprague-Dewley (SD)大鼠(雌雄各半)随机分成四组：对照组(自来水,氟含量低于1mg/L)和25mg/L.50mg/L、100mg/L三个氟化钠剂量组(氟化钠溶解于自来水中),自由饮水染毒10天后,雌、雄大鼠按1：1合笼,受孕期间继续按照上述剂量染毒至仔鼠断乳(出生后第21天),将所有雄性仔鼠按与亲代相同的染毒方式处理,到仔鼠出生后第8周末(出生后第56天)结束。应用苏木精—伊红(Hematoxylin-eosin staining, HE)染色、原位末端转移酶标记(Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, TUNEL)试验检测睾丸组织形态改变及生殖细胞凋亡情况；电镜下观察睾丸超微结构改变；采用比色法测定睾丸组织丙二醛(Methane dicarboxylic aldehyde, MDA)含量及超氧化物歧化酶(Superoxide dismutase, SOD)活力；应用Real time PCR检测内质网应激相关分子及促炎症介质的mRNA表达水平；Western blot技术检测内质网应激标志分子及核转录因子κB (nuclear factor-KB, NF-κB)的蛋白表达水平；免疫组织化学检测内质网应激凋亡调控分子生长抑制和DNA损伤同源蛋白153(GADD153/C/EBPhomologous protein, CHOP)以及炎症调控因子NF-ΚB活化亚单位p65的表达和组织定位。结果：与对照组相比,发育期氟暴露可导致明显的睾丸组织病理学损伤,主要表现为：子代大鼠睾丸曲细精管萎缩,生精上皮松弛、结构不完整,生殖细胞排列不规则,严重者甚至出现脱落,生殖细胞数量明显减少。TUNEL染色检测发现50mg/L和100mg/L染氟组睾丸出现不同程度的凋亡,主要以精原细胞和精母细胞凋亡为主。电镜结果同时也发现生殖细胞出现核染色质浓缩及边集等典型的凋亡形态学特征,并可见细胞空泡化改变；大量线粒体出现明显的肿胀、扩张,嵴断裂甚至消失等异常形态；内质网也呈极度的扩张变性改变。生化分析结果表明氟能引起睾丸组织MDA蓄积以及SOD活力降低。进一步分析发现,25mg/L、50mg/L和100mg/L氟暴露能引起睾丸内质网应激和未折叠蛋白反应活化,诱导葡萄糖调节蛋白78(Glucose-regulated protein78, GRP78)、内质网跨膜感应蛋白肌醇需求酶1(Inositol-requiring enzyme1,IRE1)及内质网凋亡信号调控蛋白CHOP的基因和蛋白表达,且CHOP的表达和组织分布与凋亡的生殖细胞大体一致。此外,25mg/L、50mg/L和100mg/L氟暴露还能不同程度的上调睾丸组织炎症相关介质如肿瘤坏死因子a(Tumor necrosis factor-α, TNF-α)、白细胞介素1β(Interleukin-1β,IL-1β)、诱导型的一氧化氮合酶(Inducible nitric oxide synthase,iNOS)和环氧合酶2(Cyclooxygenase-2, COX-2)的基因表达水平,并促进炎症转录因子NF-κBp65从胞浆到胞核的易位和活化。结论：除氧化应激外,内质网应激和炎症反应也与氟导致的精子发生障碍和生精细胞数量丢失密切相关,三者共同参与了发育期氟暴露所致的大鼠睾丸损伤过程。更多还原

【Abstract】 Part1Low glucose utilization and neurodegenerative changes caused by sodium fluoride exposure in rat’s developmental brainObjective:Fluorine, a toxic and reactive element, is widely prevalent throughout the environment and can induce toxicity when absorbed into the body. This study was to explore the possible mechanisms of developmental neurotoxicity in rats treated with different levels of sodium fluoride (NaF).Methods:Sprague-Dawley rats were administered with NaF at25,50and100mg/L via drinking water from pre-pregnancy (10days before mating) to gestation, birth and finally to post-puberty (Postnatal day56). The offspring’s learning and memory, as well as changes in neuronal morphology, glucose utilization, and functional genes expression within the brain were determined using the Morris water maze test, transmission electron microscopy (TEM), small-animal magnetic resonance imaging (MRI) and Positron emission tomography/computed tomography (PET/CT), and Western blotting techniques.Results:Fluoride treatment impaired learning and memory in offspring rats, as evidenced by increased latency time and total distance swam in the place navigation test, as well as decreased ratios (the time in the platform quadrant compared to the total time and the distance travelled in the platform quadrant compared to the total distance traveled) in the spatial probe test. In addition, MRI results showed an increase in the T2-weighted signal intensity in some areas of the lateral ventricle, suggestive of enlargement of the lateral ventricle. Also, PET/CT fusion imaging desmontrated lower glucose utilization with the standardized uptake values (SUV, fold of control) significantly reduced. Furthermore, TEM analysis revealed that fluoride caused neuronal degeneration and demyelination, together with apparent hippocampal CA1vacuolation. Functional genes analysis indicated that fluoride down-regulated the protein expression of glucose transporterl (GLUT1) and glial fibrillary acidic protein (GFAP), and up-regulated the level of brain-derived neurotrophic factor (BDNF) in both cerebral cortex and hippocampus of rat brains.Conclusions:The developmental neurotoxicity of fluoride may be closely associated with low glucose utilization and neurodegenerative changes. Part2Fluoride-elicited developmental testicular toxicity in rats:roles of endoplasmic reticulum stress and inflammatory responseObjective:Long-term excessive fluoride intake is known to be toxic and can damage a variety of organs and tissues in the human body. However, the molecular mechanisms underlying fluoride-induced male reproductive toxicity are not well understood. In this study, we used a rat model to simulate the situations of human exposure and aimed to evaluate the roles of endoplasmic reticulum (ER) stress and inflammatory response in fluoride-induced testicular injury.Methods:Sprague-Dawley rats were administered with sodium fluoride (NaF) at25,50and100mg/L via drinking water from pre-pregnancy (10days before mating) to gestation, birth and finally to post-puberty (postnatal day56). And then the testes of male offspring were collected, one part of the testis was used for histopathological and immunohistochemical analysis or Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, another part was applied for ultrastructural analysis, while the left testis was used for RNA isolation and protein extraction to determine the expression of markers for ER stress and unfolded protein response, as well as imflammatory response.Results:Our results demonstrated that fluoride treatment increased MDA accumulation, decreased SOD activity, and enhanced germ cell apoptosis. In addition, fluoride elevated mRNA and protein levels of glucose-regulated protein78(GRP78), inositol requiring ER-to-nucleus signal kinase1(IRE1), and C/EBP homologous protein (CHOP), indicating activation of ER stress signaling. Furthermore, fluoride also induced testicular inflammation, as manifested by gene up-regulation of tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX-2) and, in a nuclear factor-κB (NF-κB)-dependent manner. These were associated with marked histopathological lesions including injury of spermatogonia, decrease of spermatocytes and absence of elongated spermatids, as well as severe ultrastructural abnormalities in testes.Conclusions:Our results provide compelling evidence that ER stress and inflammation would be novel and significant mechanisms responsible for fluoride-induced disturbance of spermatogenesis and germ cell loss in addition to oxidative stress.更多还原