有機磷阻燃劑TCEP誘導(dǎo)N2a細胞的線粒體過氧化損傷
發(fā)布時間：2018-08-06 18:39
【摘要】：三-(β-氯乙基)磷酸酯(Tris(2-chloroethyl)phosphate,TCEP)作為優(yōu)良的阻燃增塑劑,廣泛用于建筑材料、家具、兒童玩具、電子產(chǎn)品和食品包裝袋等產(chǎn)品的制造過程中。TCEP作為添加劑與材料主要以非化學(xué)鍵方式結(jié)合,因此,會隨著產(chǎn)品的不斷磨損或長時間的使用而揮發(fā)到環(huán)境中,且在自然條件下極難降解。TCEP可通過空氣吸入、飲食攝入和皮膚接觸等多種途徑進入體內(nèi)。已有大量研究發(fā)現(xiàn),TCEP具有生殖毒性和發(fā)育毒性,能夠影響心臟、肝臟、腎臟和睪丸等器官的發(fā)育。近些年也有少量關(guān)于TCEP神經(jīng)毒性的報道,但有關(guān)TCEP對神經(jīng)細胞的毒性作用機制尚不清楚。大腦是機體中代謝活性最旺盛的器官,需要大量的ATP供能,線粒體通過呼吸作用氧化葡萄糖產(chǎn)生ATP,以滿足腦細胞的功能活動需求,因此線粒體的功能活動正常,對腦結(jié)構(gòu)和功能正常具有重要的意義。而線粒體在合成ATP過程中會釋放活性氧自由基(Reactive oxide species,ROS),過量的ROS可導(dǎo)致細胞過氧化損傷。已有報道TCEP可誘導(dǎo)細胞的氧化應(yīng)激相關(guān)蛋白表達異常。細胞中ROS主要產(chǎn)生于線粒體,但目前為止,尚無TCEP對神經(jīng)細胞線粒體功能影響的報道。因此,本研究以小鼠腦神經(jīng)瘤細胞系(N2a)為實驗材料,探討TCEP是否可以誘導(dǎo)線粒體的過氧化損傷及可能的機制,以及TCEP對自噬調(diào)節(jié)蛋白HDAC6表達和活性的影響,以期為TCEP的神經(jīng)毒性作用研究提供新的實驗數(shù)據(jù)。本試驗用不同濃度的TCEP (25、50、75、100 λM)分別處理N2a細胞24 hr后,通過CCK-8法檢測TCEP對細胞活性的影響;并利用DCFH-DA法、TBARS比色法和羥胺法分別檢測細胞中ROS水平、丙二醛(Malondialdehyde, MDA)含量和超氧化物歧化酶(Superoxide dismutase, SOD)活性的變化,以判斷TCEP是否對細胞造成過氧化損傷。用25或50 μM抗氧化劑維生素E (Vitamin E, VitE)預(yù)處理細胞1 hr后,再用50 μμMTCEP處理24 hr,使用DCFH-DA法檢測線粒體中ROS水平,利用JC-1技術(shù)檢測線粒體膜電位的改變,又通過比色法檢測線粒體功能的相關(guān)指標Ca2+-ATPase、細胞色素c (Cytc)氧化酶活性的變化,來判斷線粒體的過氧化損傷的可能機制;使用免疫印跡技術(shù)檢測細胞凋亡相關(guān)蛋白Bcl-2、Caspase-9、Caspase-3含量的改變,以探討線粒體依賴的細胞凋亡過程的變化;利用免疫印跡技術(shù)檢測自噬相關(guān)蛋白HDAC6和Ac-tubulin含量的改變,此外,又通過比色分析HDAC6活性的變化,采用免疫熒光標記和激光共聚焦技術(shù)觀察細胞骨架結(jié)構(gòu)的變化,來探究TCEP對自噬途徑的影響。結(jié)果發(fā)現(xiàn):(1)隨著TCEP濃度增加,細胞活性逐漸下降。(2)與對照組相比,細胞中ROS水平、MDA含量以及SOD活性呈TCEP濃度依賴性上升,但SOD與MDA的相對比值則表現(xiàn)出TCEP濃度依賴性下降的趨勢。(3)與對照組相比,隨著TCEP濃度的增加,線粒體內(nèi)ROS水平逐漸上升。(4)與對照組相比,加50 μMTCEP處理后,熒光顯微鏡下觀察發(fā)現(xiàn),發(fā)出紅色熒光信號的細胞數(shù)量明顯減少,提示線粒體膜電位下降;此外,線粒體膜上Ca2+-ATPase活性和Cytc氧化酶活性與對照組相比均顯著性下降(P0.01, P0.05)。(5)與對照組相比,50μμMTCEP處理組,抗凋亡因子Bcl-2蛋白含量顯著減少(P0.01),Caspase-9的活性片段增加,且活化的Caspase-3蛋白含量顯著性增多(P0.01)。(6)與對照組相比,50μM TCEP處理后,HDAC6的表達量沒有明顯改變。但是,HDAC6的活性顯著下調(diào)(P0.01),且HDAC6的去乙酰化底物tubulin的乙�；揎楋@著增加(P0.01)。(7)利用激光共聚焦顯微鏡觀察發(fā)現(xiàn),對照組細胞中微管成厚束狀分布,而50 μM TCEP處理后,胞內(nèi)的微管密度減少且細胞微管網(wǎng)狀結(jié)構(gòu)瓦解。(8) VitE預(yù)處理能夠阻止50 μM TCEP所引起的線粒體過氧化損傷,凋亡相關(guān)蛋白的變化,HDAC6活性的下調(diào),tubulin乙酰化修飾上調(diào)以及微管結(jié)構(gòu)的瓦解。以上結(jié)果表明:TCEP處理能夠抑制N2a細胞活性并誘導(dǎo)線粒體內(nèi)的ROS水平升高,引起線粒體過氧化損傷,進而激活細胞內(nèi)源性凋亡通路。此外,損傷的線粒體主要經(jīng)自噬通路清除,而TCEP能夠降低HDAC6活性,導(dǎo)致微管網(wǎng)狀結(jié)構(gòu)瓦解,這些異常改變可能導(dǎo)致受損線粒體經(jīng)自噬途徑的降解異常。本研究為TCEP的神經(jīng)細胞毒性研究提供了一定的實驗依據(jù)。
[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.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114
本文編號：2168636
[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.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114
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