环境激素壬基酚对小鼠神经毒性作用及其分子生物学机制研究

发布时间：2019-01-08 20:07

【摘要】： 壬基酚(nonylphenol, NP)是一种非常典型的烷基酚类化合物,被广泛应用于工农业和一些日用品的生产中,并被排放到自然界,造成环境污染。作为一种环境激素,壬基酚暴露对动物的生殖系统、免疫系统都有一定的干扰作用。然而,目前对于壬基酚的神经毒性作用的研究还很少。本论文首次通过体内实验从ROS介导的细胞凋亡以及炎症反应信号通路角度,探讨了NP的神经毒性作用及其相应的分子机制,为后续的深入研究以及防治烷基酚类环境激素神经毒性作用提供理论基础,并为制定相关环境污染标准提供理论依据。本实验采用灌胃法建立NP暴露的亚慢性毒性实验模型。将雄性小鼠随机分为4组,每组9只。处理组分别灌喂50, 100, 200 mg/kg/d的壬基酚(nonylphenol, NP)(玉米油溶解),对照组灌喂玉米油。连续处理90天之后,开始进行各种检测。利用生化实验方法,对实验小鼠中枢神经系统中的抗氧化系统进行检测,发现NP可以显著抑制抗氧化酶过氧化物歧化酶和过氧化氢酶的活性,激活单胺氧化酶的活性,而抑制小鼠脑组织的总抗氧化能力和抑制羟自由基能力,引起自由基在脑组织中的积累,引起氧化应激,造成脂质过氧化。利用基因克隆以及体外转录的方法合成探针进行原位杂交,并结合RT-PCR、免疫组化和Western Blot等分子生物学方法,对细胞凋亡信号通路上关键基因表达进行检测,最后应用TUNEL法检测细胞凋亡水平。实验发现,NP能够显著抑制脑组织中皮质和海马区域抗凋亡基因bcl-2的转录,活化Caspase-3蛋白,诱导脑组织中神经细胞凋亡。但是NP对于细胞凋亡内在通路中的促凋亡蛋白bax mRNA以及外在通路中的关键蛋白Fas和Fas-L的表达都没有影响。这提示NP诱导的中枢神经系统细胞凋亡是通过抑制bcl-2的转录并激活Caspase-3蛋白实现的。利用Western Blot和免疫组化的方法,分别对炎症反应中的关键蛋白的表达进行定量和定位检测。结果发现NP可以促进转录因子NF-κB的活化,诱导其下游炎症反应酶iNOS和COX-2在海马和皮质中的表达。同时,通过生化实验还发现总NOS活性和NO水平在NP处理组也有显著提高,提示NP具有潜在的诱导脑组织慢性炎症反应以及神经毒性的作用。通过开场行为学实验、避暗试验以及Morris水迷宫实验,检测NP对小鼠学习记忆能力以及对新异环境的兴奋性、适应性、探究和紧张程度等行为的影响,发现高剂量的NP对小鼠的空间学习记忆能力有明显的削弱作用,并使其在新异环境中适应性减弱,更容易紧张以及探究行为减少。从整体行为学角度进一步说明了NP对小鼠的中枢神经系统有一定的损伤作用。通过以上一系列的研究发现,NP对于雄性小鼠中枢神经系统有一定的毒害作用,大剂量长期接触NP对中枢神经系统会造成一定的损伤。NP诱导的神经毒性很有可能是通过促进神经细胞内ROS生成,激活线粒体依赖的细胞凋亡途径以及NF-κB介导的炎症信号通路,从而影响细胞凋亡因子以及炎症因子的表达,引起神经细胞的炎症反应促进细胞凋亡,从而对中枢神经系统起到毒性作用。
[Abstract]:Nonylphenol (NP) is a very typical alkylphenol compound, which is widely used in the production of industrial and agricultural and some daily necessities and is discharged to the natural world, causing environmental pollution. As an environmental hormone, the exposure of nonylphenol to the reproductive system and the immune system of the animal has a certain interference effect. However, there are few studies on the neurotoxicity of nonylphenol. In this paper, the neurotoxic effect of NP and the corresponding molecular mechanism of the NP were discussed for the first time in vivo, from the ROS-mediated apoptosis and the signal pathway of the inflammatory response, and a theoretical basis for the further study and the prevention and treatment of the neurotoxic effects of the alkylphenol-based environmental hormone was discussed. and provides a theoretical basis for the development of relevant environmental pollution standards. In this experiment, the subchronic toxicity of NP exposure was established by intragastric administration. Model. Male mice were randomly divided into 4 groups, each group 9. The treatment group was fed with nonylphenol (NP) of 50, 100, 200 mg/ kg/ day, respectively. The rice oil. After 90 days of continuous treatment, various kinds of oil are started. The anti-oxidation system in the central nervous system of the experimental mice was tested by biochemical experiment. It was found that NP could significantly inhibit the activity of the anti-oxidation enzyme, the superoxide dismutase and the catalase, and activate the single-amine oxygen. The activity of the enzyme is inhibited, the total antioxidant capacity of the brain tissue of the mouse is inhibited, the ability of the hydroxyl radical is inhibited, the accumulation of the free radicals in the brain tissue is caused, the oxidative stress is caused, In situ hybridization was carried out by using the method of gene cloning and in vitro transcription, and the key gene expression on the cell apoptosis signal pathway was detected by using a molecular biological method such as RT-PCR, immunohistochemistry and Western Blot, and the TUNEL method was used. It was found that NP could significantly inhibit the transcription of the anti-apoptotic gene bcl-2 in the cortex and hippocampus of the brain, activate the Caspase-3 protein, and induce the brain group. Apoptosis of the nerve cells in the weaving. However, the expression of the expression of bax mRNA and the expression of Fas and Fas-L in the pathway of apoptosis in the internal pathway of the apoptosis of the NP There is no effect on the expression of the central nervous system, which suggests that the apoptosis of the central nervous system induced by NP is by inhibiting the transcription of bcl-2 and activating Casas. e-3 protein realization. Western Blot and immunohistochemistry were used to study the key proteins in the inflammatory reaction, respectively. The results show that NP can promote the activation of transcription factor NF-B and induce the downstream inflammatory reaction enzyme iNOS and COX-2. 2 In the hippocampus and the cortex, the total NOS activity and NO level were also significantly increased in the NP treatment group, suggesting that the NP had the potential to induce the chronic inflammation of the brain tissue. The effects of NP on learning and memory of mice and the excitability and adaptability to the new environment were tested by opening behavior experiment, dodging test and Morris water maze test. The effect of high dose of NP on the learning and memory ability of mice was found to have a significant effect on the learning and memory ability of the mice, and the adaptability of the NP to the new foreign environment was weakened. It is more likely to be nervous and to reduce the inquiry behavior. The NP-to-mouse model is further explained from the overall behavioral angle. The central nervous system has a certain damage. Through the above series of studies, the NP has a certain toxic effect on the central nervous system of the male mice, and the large-dose long-term contact NPs can cause some damage to the central nervous system. NP-induced neurotoxicity is likely to be through the promotion of ROS production in the nerve cells, the activation of the mitochondrial-dependent apoptotic pathway, and the NF-B-B-mediated inflammatory signal pathway. the expression of the apoptosis factor and the inflammatory factor is affected, and the inflammatory reaction of the nerve cell is caused to promote the cell proliferation.
【学位授予单位】：中国矿业大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

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