有机磷阻燃剂TCEP诱导N2a细胞的线粒体过氧化损伤

发布时间：2018-08-06 18:39

【摘要】：三-(β-氯乙基)磷酸酯(Tris(2-chloroethyl)phosphate,TCEP)作为优良的阻燃增塑剂,广泛用于建筑材料、家具、儿童玩具、电子产品和食品包装袋等产品的制造过程中。TCEP作为添加剂与材料主要以非化学键方式结合,因此,会随着产品的不断磨损或长时间的使用而挥发到环境中,且在自然条件下极难降解。TCEP可通过空气吸入、饮食摄入和皮肤接触等多种途径进入体内。已有大量研究发现,TCEP具有生殖毒性和发育毒性,能够影响心脏、肝脏、肾脏和睾丸等器官的发育。近些年也有少量关于TCEP神经毒性的报道,但有关TCEP对神经细胞的毒性作用机制尚不清楚。大脑是机体中代谢活性最旺盛的器官,需要大量的ATP供能,线粒体通过呼吸作用氧化葡萄糖产生ATP,以满足脑细胞的功能活动需求,因此线粒体的功能活动正常,对脑结构和功能正常具有重要的意义。而线粒体在合成ATP过程中会释放活性氧自由基(Reactive oxide species,ROS),过量的ROS可导致细胞过氧化损伤。已有报道TCEP可诱导细胞的氧化应激相关蛋白表达异常。细胞中ROS主要产生于线粒体,但目前为止,尚无TCEP对神经细胞线粒体功能影响的报道。因此,本研究以小鼠脑神经瘤细胞系(N2a)为实验材料,探讨TCEP是否可以诱导线粒体的过氧化损伤及可能的机制,以及TCEP对自噬调节蛋白HDAC6表达和活性的影响,以期为TCEP的神经毒性作用研究提供新的实验数据。本试验用不同浓度的TCEP (25、50、75、100 λM)分别处理N2a细胞24 hr后,通过CCK-8法检测TCEP对细胞活性的影响;并利用DCFH-DA法、TBARS比色法和羟胺法分别检测细胞中ROS水平、丙二醛(Malondialdehyde, MDA)含量和超氧化物歧化酶(Superoxide dismutase, SOD)活性的变化,以判断TCEP是否对细胞造成过氧化损伤。用25或50 μM抗氧化剂维生素E (Vitamin E, VitE)预处理细胞1 hr后,再用50 μμMTCEP处理24 hr,使用DCFH-DA法检测线粒体中ROS水平,利用JC-1技术检测线粒体膜电位的改变,又通过比色法检测线粒体功能的相关指标Ca2+-ATPase、细胞色素c (Cytc)氧化酶活性的变化,来判断线粒体的过氧化损伤的可能机制;使用免疫印迹技术检测细胞凋亡相关蛋白Bcl-2、Caspase-9、Caspase-3含量的改变,以探讨线粒体依赖的细胞凋亡过程的变化;利用免疫印迹技术检测自噬相关蛋白HDAC6和Ac-tubulin含量的改变,此外,又通过比色分析HDAC6活性的变化,采用免疫荧光标记和激光共聚焦技术观察细胞骨架结构的变化,来探究TCEP对自噬途径的影响。结果发现:(1)随着TCEP浓度增加,细胞活性逐渐下降。(2)与对照组相比,细胞中ROS水平、MDA含量以及SOD活性呈TCEP浓度依赖性上升,但SOD与MDA的相对比值则表现出TCEP浓度依赖性下降的趋势。(3)与对照组相比,随着TCEP浓度的增加,线粒体内ROS水平逐渐上升。(4)与对照组相比,加50 μMTCEP处理后,荧光显微镜下观察发现,发出红色荧光信号的细胞数量明显减少,提示线粒体膜电位下降;此外,线粒体膜上Ca2+-ATPase活性和Cytc氧化酶活性与对照组相比均显著性下降(P0.01, P0.05)。(5)与对照组相比,50μμMTCEP处理组,抗凋亡因子Bcl-2蛋白含量显著减少(P0.01),Caspase-9的活性片段增加,且活化的Caspase-3蛋白含量显著性增多(P0.01)。(6)与对照组相比,50μM TCEP处理后,HDAC6的表达量没有明显改变。但是,HDAC6的活性显著下调(P0.01),且HDAC6的去乙酰化底物tubulin的乙酰化修饰显著增加(P0.01)。(7)利用激光共聚焦显微镜观察发现,对照组细胞中微管成厚束状分布,而50 μM TCEP处理后,胞内的微管密度减少且细胞微管网状结构瓦解。(8) VitE预处理能够阻止50 μM TCEP所引起的线粒体过氧化损伤,凋亡相关蛋白的变化,HDAC6活性的下调,tubulin乙酰化修饰上调以及微管结构的瓦解。以上结果表明:TCEP处理能够抑制N2a细胞活性并诱导线粒体内的ROS水平升高,引起线粒体过氧化损伤,进而激活细胞内源性凋亡通路。此外,损伤的线粒体主要经自噬通路清除,而TCEP能够降低HDAC6活性,导致微管网状结构瓦解,这些异常改变可能导致受损线粒体经自噬途径的降解异常。本研究为TCEP的神经细胞毒性研究提供了一定的实验依据。
[Abstract]:Three - (beta chloroethyl) phosphate (Tris (2-chloroethyl) phosphate, TCEP), as a fine flame retardant plasticizer, is widely used in the manufacturing process of building materials, furniture, children's toys, electronic products and food packaging bags, and.TCEP as additives and materials is mainly combined with non chemical bonds, as a result, with the continuous wear or tear of the products. Long time use volatilizes into the environment and is extremely difficult to degrade under natural conditions..TCEP can enter into the body through air inhalation, dietary intake and skin contact. A large number of studies have found that TCEP has reproductive toxicity and developmental toxicity, which can affect the development of heart, liver, kidney and testis. It is not clear about the TCEP neurotoxicity, but the mechanism of the toxic effect of TCEP on nerve cells is not clear. The brain is the most potent metabolic organ in the body and requires a large number of ATP for energy. Mitochondria oxidize glucose to produce ATP to meet the functional activity needs of the brain cells, so the function of mitochondria is normal, It is of great significance to the normal brain structure and function. While mitochondria release Reactive oxide species (ROS) in the process of synthesis of ATP, excessive ROS can cause oxidative damage to cells. It has been reported that TCEP can induce the abnormal expression of oxidative stress related proteins in cells. In cells, ROS is mainly produced in mitochondria, but is currently in the mitochondria. So far, there is no report on the effect of TCEP on the function of mitochondrial function of mitochondria. Therefore, this study uses the mouse brain neuroma cell line (N2a) as an experimental material to explore whether TCEP can induce mitochondrial oxidative damage and possible mechanisms, as well as the effect of TCEP on the expression and activity of autophagic regulatory protein HDAC6, in order to be the neurotoxic effect of TCEP. The study provided new experimental data. After treating N2a cells 24 hr with different concentrations of TCEP (25,50,75100 lambda M), the effect of TCEP on cell activity was detected by CCK-8, and DCFH-DA method, TBARS colorimetric assay and hydroxylamine method were used to detect the level of ROS, the content of propylene two aldehyde (Malondialdehyde, MDA) and superoxide dismutase (SOD). Superoxide dismutase, SOD) activity changes to determine whether TCEP causes oxidative damage to cells. After the pretreatment of 1 hr with 25 or 50 mu M antioxidant vitamin E (Vitamin E, VitE), the 24 hr is treated with 50 mu MTCEP. Detection of mitochondrial function related index Ca2+-ATPase and cytochrome c (Cytc) oxidase activity to determine the possible mechanism of mitochondrial oxidative damage, and the changes of apoptosis related protein Bcl-2, Caspase-9, Caspase-3 content using Western blot, in order to explore the apoptosis process of mitochondria dependent cells. The changes in the content of autophagy related proteins HDAC6 and Ac-tubulin were detected by immunoblotting. In addition, the changes in the activity of HDAC6 in the colorimetric analysis were also observed. The changes in the cytoskeleton structure were observed by immunofluorescence markers and laser confocal technology to explore the effect of TCEP on the autophagy pathway. The results were as follows: (1) with the increase of TCEP concentration Cell activity decreased gradually. (2) compared with the control group, ROS level, MDA content and SOD activity increased in TCEP concentration, but the relative ratio of SOD and MDA showed a tendency to decrease TCEP concentration dependence. (3) compared with the control group, the level of ROS increased gradually with the increase of TCEP concentration. (4) compared with the control group, After 50 mu MTCEP treatment, it was observed under the fluorescence microscope that the number of cells emitting red fluorescent signal decreased significantly, suggesting the decrease of mitochondrial membrane potential. In addition, the Ca2+-ATPase activity and Cytc oxidase activity on the membrane of the mitochondria decreased significantly (P0.01, P0.05) compared with the control group. (5) compared with the control group, the 50 UU MTCEP treatment group was resistant to withering. The Bcl-2 protein content of the dead factor decreased significantly (P0.01), the active fragment of Caspase-9 increased and the activated Caspase-3 protein content increased significantly (P0.01). (6) the expression of HDAC6 was not significantly changed after 50 M TCEP treatment. However, the activity of HDAC6 decreased significantly (P0.01), and the HDAC6 acetylated substrate tubulin (7) (7) the microtubules in the control group were found to be thick and fascicular in the control group by laser confocal microscopy, and the microtubule density in the cell was reduced and the cell microtubule reticular formation was disintegrated after 50 micron TCEP treatment. (8) the VitE pretreatment could prevent the mitochondrial peroxidation damage caused by 50 mu M TCEP and the apoptosis related protein. The changes, the down regulation of HDAC6 activity, the up-regulation of tubulin acetylation modification and the disintegration of microtubule structure. The results showed that TCEP treatment could inhibit the activity of N2a cells and induce the increase of ROS levels in mitochondria, causing mitochondrial oxidative damage and activating the endogenous apoptosis pathway of the cells. In addition, the damaged mitochondria were mainly autophagy through autophagy. TCEP can reduce the activity of HDAC6 and cause the disintegration of microtubule reticular structure. These abnormal changes may lead to abnormal degradation of the damaged mitochondria through autophagy pathway. This study provides some experimental basis for the study of the neurotoxicity of TCEP.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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