本文選題：芒果苷　切入點(diǎn)：鉛　出處：《華中科技大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：芒果苷是一種高效的抗氧化劑，且具有絡(luò)合、吸附及還原重金屬的功能，這些性質(zhì)使其緩解重金屬離子的毒害作用成為可能。 鉛是常見的環(huán)境污染物，可通過多種途徑進(jìn)入體內(nèi)，造成機(jī)體多種器官系統(tǒng)的損害，中樞神經(jīng)系統(tǒng)是毒性作用的主要靶器官之一，兒童對鉛毒性尤為敏感，較低水平的鉛暴露即可造成中樞神經(jīng)系統(tǒng)功能障礙。已有研究表明，活性氧介導(dǎo)的氧化損傷參與鉛中毒的病理學(xué)過程。近年發(fā)現(xiàn)的紅系衍生核因子2相關(guān)因子和抗氧化反應(yīng)元件（Nrf2-ARE）通路具有神經(jīng)保護(hù)作用。 本研究以鉛引起神經(jīng)組織氧化應(yīng)激為切入點(diǎn)，利用整體動物實(shí)驗(yàn)?zāi)Ｐ�，�?yīng)用生物化學(xué)與分子細(xì)胞生物學(xué)技術(shù)和方法來研究：①芒果苷對斷乳期鉛暴露大鼠的空間學(xué)習(xí)記憶能力以及大腦皮層、海馬組織結(jié)構(gòu)的影響；對鉛暴露大鼠體內(nèi)鉛負(fù)荷的影響；②芒果苷是否通過誘導(dǎo)Nrf2-ARE通路下游的相關(guān)酶參與改善鉛暴露大鼠的氧化損傷；③Nrf2-ARE通路是否在芒果苷拮抗鉛致大鼠腦損害中發(fā)揮作用。主要研究結(jié)果如下： 第一部分芒果苷對鉛暴露大鼠神經(jīng)系統(tǒng)結(jié)構(gòu)、功能及體內(nèi)鉛負(fù)荷的影響 目的：研究芒果苷對鉛暴露大鼠神經(jīng)系統(tǒng)結(jié)構(gòu)、功能及體內(nèi)鉛負(fù)荷的影響。 方法：將96只Wistar大鼠隨機(jī)分為陰性對照組(空白對照組)和鉛暴露組(以500ppm醋酸鉛溶液作為大鼠飲用水)，染毒8周后，將鉛暴露后的大鼠再隨機(jī)分為5個組，以50、100和200mg/kg劑量的芒果苷（藥物組）和二巰基丁二酸（DMSA）組分別給其中四組大鼠灌胃，剩余一組(鉛暴露模型組)給予等體積蒸餾水。給予芒果苷4周后，用Morris水迷宮進(jìn)行空間學(xué)習(xí)記憶能力檢測。Morris水迷宮實(shí)驗(yàn)結(jié)束后，脫頸椎處死動物，采用電感耦合等離子體質(zhì)譜法(Inductively coupled plasma massspectrometry,ICP-MS)進(jìn)行鉛含量測定。HE染色，光鏡下觀察大腦皮層病理改變，TEM透射電鏡觀察海馬CA1區(qū)超微結(jié)構(gòu)。 結(jié)果：Morris水迷宮實(shí)驗(yàn)中，鉛暴露模型組與空白模型組相比，各統(tǒng)計(jì)指標(biāo)沒有統(tǒng)計(jì)學(xué)差異。芒果苷治療組(200mg/kg)與鉛暴露模型組比，第三象限的停留時間較長，跨平臺次數(shù)較多，均有顯著性差異(p 0.05)。鉛暴露對斷乳期大鼠體重影響不明顯，但可引起海馬組織內(nèi)各種細(xì)胞超微病理結(jié)構(gòu)的變化，包括空泡化、線粒體腫脹、核濃縮和凋亡等，芒果苷治療組(100，200mg/kg)上述病理改變有很大改觀。大鼠斷乳期鉛暴露，可使大鼠血液及各臟器中鉛含量增加，芒果苷能使鉛負(fù)荷降低。其在骨和腦中的效果與DMSA組相比沒有顯著性差異。 結(jié)論：鉛對本實(shí)驗(yàn)中斷乳期大鼠空間學(xué)習(xí)記憶能力沒有明顯影響，這可能和大鼠的神經(jīng)代償有關(guān)；和空白組相比，芒果苷治療組(200mg/kg)能顯著提高大鼠的空間學(xué)習(xí)記憶能力；芒果苷能改善斷乳期鉛暴露大鼠的病理損害，對鉛暴露大鼠有保護(hù)作用；芒果苷能降低血液及骨、腦、肝和腎鉛，，這可能和它的螯合特性有關(guān)；芒果苷能降低腦鉛，可能與它的分子量較小、較易穿透血腦屏障有關(guān)。 第二部分對血液和腦組織的氧化損傷的保護(hù)作用 目的：研究芒果苷是否干預(yù)Nrf2-ARE通路所調(diào)控的抗氧化酶、II相解毒酶、谷胱甘肽及相關(guān)的調(diào)節(jié)酶。 方法：應(yīng)該商用試劑盒檢測H2O2、MDA含量以及Nrf2下游的抗氧化酶（SOD，CAT）活力、II相代謝酶(GST，NQO1，HO-1)活力、谷胱甘肽（GSH）調(diào)節(jié)酶類(γ-GCS，GR，GPx)以及GSH和GSSG的含量。 結(jié)果：鉛可以顯著提高脂質(zhì)過氧化物水平，降低抗氧化物酶活力。不同濃度的芒果苷治療組可以顯著降低脂質(zhì)過氧化物水平，提高抗氧化物酶活力，其中200mg/kg芒果苷的作用最為明顯。鉛可以顯著抑制HO-1、NQO1酶，同時也抑制了GSH相關(guān)調(diào)節(jié)酶，GSH耗竭，GSH/GSSG比例下降。芒果苷治療組可以提高II相代謝酶以及谷胱甘肽調(diào)節(jié)酶類，提高GSH含量和GSH/GSSG比例，其中200mg/kg芒果苷的作用最為明顯。 結(jié)論：芒果苷治療各組可以提高機(jī)體氧化還原能力，提高大腦組織及血中Nrf2下游的II相代謝酶以及GSH調(diào)節(jié)酶類活力，抑制由鉛誘導(dǎo)的氧化壓力，從而拮抗鉛誘導(dǎo)的損傷。以上研究結(jié)果提示Nrf2-ARE通路可能參與了芒果苷的氧化應(yīng)激防御機(jī)制。 第三部分Nrf2-ARE信號通路在芒果苷拮抗鉛致大鼠腦損害中的作用 目的：研究Nrf2-ARE通路是否在芒果苷拮抗鉛致大鼠腦損害中發(fā)揮作用。 方法：實(shí)時熒光定量PCR (Real-time quantitative Polymerase Chain Reaction，RT-qPCR)、Western Blot，免疫組織化學(xué)檢測Nrf2、GCLM、GCLC以及HO-1mRNA和蛋白表達(dá)；Western-blot檢測Nrf2總蛋白和Nrf2核蛋白表達(dá)。 結(jié)果：RT-qPCR表明Nrf2mRNA水平在鉛暴露大鼠有較弱的提高，在芒果苷治療各組也呈較弱的提高。γ-GCS和HO-1在鉛暴露大鼠中被抑制，在芒果苷治療各組中有顯著的提高，且呈劑量關(guān)系。免疫組化檢測顯示Nrf2在空白組未陽性神經(jīng)細(xì)胞，在鉛暴露大鼠中陽性神經(jīng)細(xì)胞少量表達(dá)，在芒果苷治療各組中表達(dá)大幅增加；γ-GCS陽性細(xì)胞在鉛暴露大鼠中較空白組少，在芒果苷治療各組中表達(dá)大幅增加。 結(jié)論：Nrf2可被鉛激活，芒果苷可以進(jìn)一步激活它，其調(diào)控并非發(fā)生于基因轉(zhuǎn)錄水平，而可能發(fā)生在轉(zhuǎn)錄后的Nrf2入核和出核轉(zhuǎn)運(yùn)水平。Nrf2可能是芒果苷干預(yù)鉛暴露大鼠抗氧化基因表達(dá)的關(guān)鍵轉(zhuǎn)錄調(diào)控因子。給予芒果苷后Nrf2的激活上調(diào)了其下游的γ-GCS、HO-1水平，提示Nrf2-ARE通路可能參與了芒果苷的氧化應(yīng)激防御機(jī)制。 綜上所述，我們可以得出如下結(jié)論：芒果苷能減輕鉛暴露大鼠的氧化損傷，對鉛暴露大鼠有神經(jīng)保護(hù)作用；這種神經(jīng)保護(hù)作用可能是通過激活Nrf2-ARE通路誘導(dǎo)下游抗氧化/解毒酶等基因的表達(dá)從而抑制氧化損傷來實(shí)現(xiàn)的；以Nrf2-ARE通路的藥物治療對鉛中毒大鼠的防治具有良好的應(yīng)用前景。Nrf2/ARE信號通路示意圖及芒果苷的可能作用機(jī)制見圖1。 本研究的創(chuàng)新之處：將強(qiáng)抗氧化劑芒果苷應(yīng)用于重金屬鉛的研究中；從Nrf2通路解釋芒果苷對神經(jīng)系統(tǒng)的保護(hù)作用。
[Abstract]:Mangiferin is an efficient antioxidant, and has the functions of complexation, adsorption and reduction of heavy metals. These properties make it possible to alleviate the toxic effect of heavy metal ions.
The lead is a common environmental pollutant, can enter the body through a variety of ways, causing the body multiple organ system damage, central nervous system is one of the main target organ toxicity, children are particularly sensitive to the toxicity of lead, low level lead exposure can cause central nervous system dysfunction. Studies have shown that the process is mediated by reactive oxygen species oxidative damage is involved in lead poisoning pathology. Recently found NF-E2 related factor 2 and antioxidant response element (Nrf2-ARE) pathway has a neuroprotective effect.
In this study, the lead induced neural tissue oxidative stress as the starting point, the use of the whole animal experiment model, molecular cell biology technology and methods of Biochemistry and research: Mangiferin on weaning lead exposed rats during spatial learning and memory ability and cerebral cortex, hippocampus and the influence of organizational structure; effects of lead exposure lead load in vivo; whether the mangiferin by related enzymes induced by Nrf2-ARE pathway downstream of the lead exposure in improving oxidative damage of rat; whether the role of Nrf2-ARE pathway in rat brain damage induced by Mangiferin on lead. The main results are as follows:
The effects of Mangiferin on neural structure, function and lead load in rats exposed to lead exposure
Objective: To study the effects of Mangiferin on the structure, function and lead load of the nervous system in rats exposed to lead.
Methods: 96 Wistar rats were randomly divided into negative control group (control group) and Pb group (with 500ppm lead acetate solution as the rats drinking water), after 8 weeks, the lead exposure rats were randomly divided into 5 groups, with 50100 doses of mangiferin and 200mg/kg (drug group) and two mercapto succinic acid (DMSA) respectively to the four groups of rats by gavage, the remaining group (lead exposure group) given equal volume of distilled water. Given the mangiferin after 4 weeks, with the Morris water maze over the ability of spatial learning and memory test of.Morris water maze experiment after removal the cervical executed animal, by inductively coupled plasma mass spectrometry (Inductively coupled plasma massspectrometry, ICP-MS) were determined by.HE staining lead content, pathological changes in the cerebral cortex were observed under light microscope to observe the ultrastructure of hippocampal CA1 area TEM transmission electron microscope.
Results: the Morris water maze test, lead exposure model group and blank model group, no statistically significant differences between the various statistical indicators. Mangiferin treatment group (200mg/kg) and lead exposure model group, the longer residence time of third quadrant, cross platform number more, there were significant difference (P 0.05). The effects of lead exposure the weight of weaning rats is not obvious, but it can cause various changes of cell ultrastructure in the hippocampus, including vacuolization, mitochondrial swelling, nuclear condensation and apoptosis, mangiferin treatment group (100200mg/kg) the pathological changes have very big change. Rats in weaning period of lead exposure, the lead content in rats blood and viscera increased, mangiferin can lead to reduce the load. The bone and brain effect compared with the DMSA group had no significant difference.
Conclusion: lead to the experiment space interrupt lactation rats did not significantly affect the ability of learning and memory, and this may be nervous compensatory rats; compared with the blank group, mangiferin treatment group (200mg/kg) can significantly improve the spatial learning and memory ability in rats; mangiferin can improve the milk off the lead exposure during pathological damage of large rats exposed to lead, has a protective effect on rat; mangiferin can reduce the blood and bone, brain, liver and kidney lead, probably related to its chelating properties; mangiferin can reduce brain lead, and its possible molecular size is small, easy to penetrate the blood-brain barrier is closed.
The second part protects the oxidative damage of blood and brain tissue
Objective: To investigate whether mangiferin interfered with antioxidant enzymes, II detoxification enzymes, glutathione and related regulatory enzymes regulated by Nrf2-ARE pathway.
Methods: the content of H2O2, MDA and Nrf2 downstream antioxidant enzyme (SOD, CAT) activity, II phase metabolizing enzyme (GST, NQO1, HO-1) activity, glutathione (GSH) regulating enzymes (gamma -GCS, HO-1), and the content of "Yu He" should be detected by commercial kits.
Results: lead can significantly improve the level of lipid peroxidation, reduce antioxidant enzyme activity. Different concentrations of mangiferin in the treatment group can significantly reduce the level of lipid peroxidation, increase the antioxidant enzyme activity, of which 200mg/kg mangiferin effect is most obvious. Lead can significantly inhibit HO-1, NQO1 enzyme, but also inhibit the enzyme related regulation of GSH GSH depletion decreased GSH/GSSG ratio. Mangiferin treatment group can improve the phase II metabolic enzymes and glutathione regulating enzymes, increasing the content of GSH and the ratio of GSH/GSSG, the 200mg/kg of mangiferin effect is most obvious.
Conclusion: mangiferin treatment groups can improve the body redox ability, improve brain tissue and blood in the downstream of the Nrf2 phase II metabolic enzymes and GSH regulating enzymes activity, inhibition of oxidative stress induced by lead, lead to antagonism induced injury. The above results suggest that oxidative stress defense mechanism of Nrf2-ARE pathway may be involved in the mangiferin.
The role of the third part of Nrf2-ARE signaling pathway in the antagonism of mangiferin to lead induced brain damage in rats
Objective: To investigate whether the Nrf2-ARE pathway plays a role in the antagonism of mangiferin to lead induced brain damage in rats.
Methods: Real-time quantitative Polymerase Chain Reaction (RT-qPCR), Western Blot, immunohistochemistry were used to detect Nrf2, GCLM, GCLC and protein expression of PCR and quantitative.
Results: RT-qPCR showed that the level of Nrf2mRNA in lead exposed rats are weaker in the treatment groups improved, mangiferin was also improved. The weak gamma -GCS and HO-1 exposure was inhibited in rats in the lead, has improved significantly in the treatment of mangiferin in each group, and there was a dose relationship. Immunohistochemical detection showed in Nrf2 blank group the positive cells in a small amount of positive expression of nerve cells in rats exposed to lead, a substantial increase in the expression of mangiferin in treatment group; gamma -GCS positive cells in the lead exposure less than the blank group rats, significantly increased expression in the treatment of mangiferin groups.
Conclusion: Nrf2 can lead activation, mangiferin can further activate it, the regulation is not on the level of gene transcription, which may occur in the post transcriptional Nrf2 into the nucleus and nuclear translocation of.Nrf2 level may be mangiferin intervention lead exposure key transcription regulation factor of antioxidant gene expression in rats. Given the activation of Nrf2 in Mango in the downstream of the -GCS after gamma, HO-1 level, oxidative stress defense mechanisms suggest that Nrf2-ARE pathway may be involved in the mangiferin.
In summary, we can draw the following conclusion: mangiferin can alleviate the oxidative damage of lead exposure in rats, rats exposed to lead a neuroprotective effect; this neuroprotective effect may be achieved through activation of Nrf2-ARE pathway induced expression of downstream antioxidant / detoxification enzyme gene to inhibit oxidative damage; Nrf2-ARE pathway to drug treatment control in lead poisoning rats with the possible mechanism of figure good application prospect of.Nrf2/ARE signal pathway and schematic diagram of mangiferin 1.
The innovation of this study is to apply the strong antioxidant mangiferin in the study of heavy metal lead, and to explain the protective effect of Mangiferin on the nervous system from the Nrf2 pathway.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R114

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 李好文,鄧家剛,鄧靜;芒果苷國外研究進(jìn)展[J];廣西中醫(yī)學(xué)院學(xué)報;2003年04期

2 袁寶珊,吳宜群;環(huán)境鉛污染與兒童健康[J];國外醫(yī)學(xué)(衛(wèi)生學(xué)分冊);1998年04期

3 黃華藝,鐘鳴,農(nóng)朝贊,趙世元,孟剛;脂質(zhì)過氧化大鼠腦組織超微結(jié)構(gòu)改變及gg果甙對腦組織的保護(hù)作用[J];廣西科學(xué);2000年02期

4 宋亞頎;王漢東;;轉(zhuǎn)錄因子NF-E2相關(guān)因子2-抗氧化轉(zhuǎn)錄元件信號路徑細(xì)胞保護(hù)作用的研究進(jìn)展[J];醫(yī)學(xué)研究生學(xué)報;2009年04期

5 王笑亮;王漢東;;Nrf2在中樞神經(jīng)系統(tǒng)疾病中的神經(jīng)保護(hù)作用[J];醫(yī)學(xué)研究生學(xué)報;2011年07期

6 龐澍華;;利用廢輪胎非法煉油是環(huán)境污染的高增長源[J];資源再生;2007年01期

7 夏小俊,金中初;NQO1酶及其被氧環(huán)境誘導(dǎo)表達(dá)的研究進(jìn)展[J];生理科學(xué)進(jìn)展;2002年03期

8 朱鳳臣;蔣電明;;Nrf2/ARE轉(zhuǎn)導(dǎo)通路在中樞神經(jīng)系統(tǒng)疾病中作用的研究進(jìn)展[J];中國生物制品學(xué)雜志;2011年06期

9 林曉萍;李雯;沈華浩;;抗氧化應(yīng)激轉(zhuǎn)錄因子-Nrf2的研究進(jìn)展[J];中國病理生理雜志;2011年06期

本文編號：1595871