環(huán)境激素壬基酚對(duì)小鼠神經(jīng)毒性作用及其分子生物學(xué)機(jī)制研究
發(fā)布時(shí)間：2019-01-08 20:07
【摘要】： 壬基酚(nonylphenol, NP)是一種非常典型的烷基酚類化合物,被廣泛應(yīng)用于工農(nóng)業(yè)和一些日用品的生產(chǎn)中,并被排放到自然界,造成環(huán)境污染。作為一種環(huán)境激素,壬基酚暴露對(duì)動(dòng)物的生殖系統(tǒng)、免疫系統(tǒng)都有一定的干擾作用。然而,目前對(duì)于壬基酚的神經(jīng)毒性作用的研究還很少。本論文首次通過(guò)體內(nèi)實(shí)驗(yàn)從ROS介導(dǎo)的細(xì)胞凋亡以及炎癥反應(yīng)信號(hào)通路角度,探討了NP的神經(jīng)毒性作用及其相應(yīng)的分子機(jī)制,為后續(xù)的深入研究以及防治烷基酚類環(huán)境激素神經(jīng)毒性作用提供理論基礎(chǔ),并為制定相關(guān)環(huán)境污染標(biāo)準(zhǔn)提供理論依據(jù)。 本實(shí)驗(yàn)采用灌胃法建立NP暴露的亞慢性毒性實(shí)驗(yàn)?zāi)Ｐ�。將雄性小鼠隨機(jī)分為4組,每組9只。處理組分別灌喂50, 100, 200 mg/kg/d的壬基酚(nonylphenol, NP)(玉米油溶解),對(duì)照組灌喂玉米油。連續(xù)處理90天之后,開(kāi)始進(jìn)行各種檢測(cè)。 利用生化實(shí)驗(yàn)方法,對(duì)實(shí)驗(yàn)小鼠中樞神經(jīng)系統(tǒng)中的抗氧化系統(tǒng)進(jìn)行檢測(cè),發(fā)現(xiàn)NP可以顯著抑制抗氧化酶過(guò)氧化物歧化酶和過(guò)氧化氫酶的活性,激活單胺氧化酶的活性,而抑制小鼠腦組織的總抗氧化能力和抑制羥自由基能力,引起自由基在腦組織中的積累,引起氧化應(yīng)激,造成脂質(zhì)過(guò)氧化。 利用基因克隆以及體外轉(zhuǎn)錄的方法合成探針進(jìn)行原位雜交,并結(jié)合RT-PCR、免疫組化和Western Blot等分子生物學(xué)方法,對(duì)細(xì)胞凋亡信號(hào)通路上關(guān)鍵基因表達(dá)進(jìn)行檢測(cè),最后應(yīng)用TUNEL法檢測(cè)細(xì)胞凋亡水平。實(shí)驗(yàn)發(fā)現(xiàn),NP能夠顯著抑制腦組織中皮質(zhì)和海馬區(qū)域抗凋亡基因bcl-2的轉(zhuǎn)錄,活化Caspase-3蛋白,誘導(dǎo)腦組織中神經(jīng)細(xì)胞凋亡。但是NP對(duì)于細(xì)胞凋亡內(nèi)在通路中的促凋亡蛋白bax mRNA以及外在通路中的關(guān)鍵蛋白Fas和Fas-L的表達(dá)都沒(méi)有影響。這提示NP誘導(dǎo)的中樞神經(jīng)系統(tǒng)細(xì)胞凋亡是通過(guò)抑制bcl-2的轉(zhuǎn)錄并激活Caspase-3蛋白實(shí)現(xiàn)的。 利用Western Blot和免疫組化的方法,分別對(duì)炎癥反應(yīng)中的關(guān)鍵蛋白的表達(dá)進(jìn)行定量和定位檢測(cè)。結(jié)果發(fā)現(xiàn)NP可以促進(jìn)轉(zhuǎn)錄因子NF-κB的活化,誘導(dǎo)其下游炎癥反應(yīng)酶iNOS和COX-2在海馬和皮質(zhì)中的表達(dá)。同時(shí),通過(guò)生化實(shí)驗(yàn)還發(fā)現(xiàn)總NOS活性和NO水平在NP處理組也有顯著提高,提示NP具有潛在的誘導(dǎo)腦組織慢性炎癥反應(yīng)以及神經(jīng)毒性的作用。 通過(guò)開(kāi)場(chǎng)行為學(xué)實(shí)驗(yàn)、避暗試驗(yàn)以及Morris水迷宮實(shí)驗(yàn),檢測(cè)NP對(duì)小鼠學(xué)習(xí)記憶能力以及對(duì)新異環(huán)境的興奮性、適應(yīng)性、探究和緊張程度等行為的影響,發(fā)現(xiàn)高劑量的NP對(duì)小鼠的空間學(xué)習(xí)記憶能力有明顯的削弱作用,并使其在新異環(huán)境中適應(yīng)性減弱,更容易緊張以及探究行為減少。從整體行為學(xué)角度進(jìn)一步說(shuō)明了NP對(duì)小鼠的中樞神經(jīng)系統(tǒng)有一定的損傷作用。 通過(guò)以上一系列的研究發(fā)現(xiàn),NP對(duì)于雄性小鼠中樞神經(jīng)系統(tǒng)有一定的毒害作用,大劑量長(zhǎng)期接觸NP對(duì)中樞神經(jīng)系統(tǒng)會(huì)造成一定的損傷。NP誘導(dǎo)的神經(jīng)毒性很有可能是通過(guò)促進(jìn)神經(jīng)細(xì)胞內(nèi)ROS生成,激活線粒體依賴的細(xì)胞凋亡途徑以及NF-κB介導(dǎo)的炎癥信號(hào)通路,從而影響細(xì)胞凋亡因子以及炎癥因子的表達(dá),引起神經(jīng)細(xì)胞的炎癥反應(yīng)促進(jìn)細(xì)胞凋亡,從而對(duì)中樞神經(jīng)系統(tǒng)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.
【學(xué)位授予單位】：中國(guó)礦業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363
[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.
【學(xué)位授予單位】：中國(guó)礦業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363
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