芒果苷对铅暴露大鼠的保护作用及信号转导机制研究

发布时间：2018-03-11 01:04

本文选题：芒果苷　切入点：铅　出处：《华中科技大学》2013年博士论文　论文类型：学位论文

【摘要】：芒果苷是一种高效的抗氧化剂，且具有络合、吸附及还原重金属的功能，这些性质使其缓解重金属离子的毒害作用成为可能。铅是常见的环境污染物，可通过多种途径进入体内，造成机体多种器官系统的损害，中枢神经系统是毒性作用的主要靶器官之一，儿童对铅毒性尤为敏感，较低水平的铅暴露即可造成中枢神经系统功能障碍。已有研究表明，活性氧介导的氧化损伤参与铅中毒的病理学过程。近年发现的红系衍生核因子2相关因子和抗氧化反应元件（Nrf2-ARE）通路具有神经保护作用。本研究以铅引起神经组织氧化应激为切入点，利用整体动物实验模型，应用生物化学与分子细胞生物学技术和方法来研究：①芒果苷对断乳期铅暴露大鼠的空间学习记忆能力以及大脑皮层、海马组织结构的影响；对铅暴露大鼠体内铅负荷的影响；②芒果苷是否通过诱导Nrf2-ARE通路下游的相关酶参与改善铅暴露大鼠的氧化损伤；③Nrf2-ARE通路是否在芒果苷拮抗铅致大鼠脑损害中发挥作用。主要研究结果如下：第一部分芒果苷对铅暴露大鼠神经系统结构、功能及体内铅负荷的影响目的：研究芒果苷对铅暴露大鼠神经系统结构、功能及体内铅负荷的影响。方法：将96只Wistar大鼠随机分为阴性对照组(空白对照组)和铅暴露组(以500ppm醋酸铅溶液作为大鼠饮用水)，染毒8周后，将铅暴露后的大鼠再随机分为5个组，以50、100和200mg/kg剂量的芒果苷（药物组）和二巯基丁二酸（DMSA）组分别给其中四组大鼠灌胃，剩余一组(铅暴露模型组)给予等体积蒸馏水。给予芒果苷4周后，用Morris水迷宫进行空间学习记忆能力检测。Morris水迷宫实验结束后，脱颈椎处死动物，采用电感耦合等离子体质谱法(Inductively coupled plasma massspectrometry,ICP-MS)进行铅含量测定。HE染色，光镜下观察大脑皮层病理改变，TEM透射电镜观察海马CA1区超微结构。结果：Morris水迷宫实验中，铅暴露模型组与空白模型组相比，各统计指标没有统计学差异。芒果苷治疗组(200mg/kg)与铅暴露模型组比，第三象限的停留时间较长，跨平台次数较多，均有显著性差异(p 0.05)。铅暴露对断乳期大鼠体重影响不明显，但可引起海马组织内各种细胞超微病理结构的变化，包括空泡化、线粒体肿胀、核浓缩和凋亡等，芒果苷治疗组(100，200mg/kg)上述病理改变有很大改观。大鼠断乳期铅暴露，可使大鼠血液及各脏器中铅含量增加，芒果苷能使铅负荷降低。其在骨和脑中的效果与DMSA组相比没有显著性差异。结论：铅对本实验中断乳期大鼠空间学习记忆能力没有明显影响，这可能和大鼠的神经代偿有关；和空白组相比，芒果苷治疗组(200mg/kg)能显著提高大鼠的空间学习记忆能力；芒果苷能改善断乳期铅暴露大鼠的病理损害，对铅暴露大鼠有保护作用；芒果苷能降低血液及骨、脑、肝和肾铅，，这可能和它的螯合特性有关；芒果苷能降低脑铅，可能与它的分子量较小、较易穿透血脑屏障有关。第二部分对血液和脑组织的氧化损伤的保护作用目的：研究芒果苷是否干预Nrf2-ARE通路所调控的抗氧化酶、II相解毒酶、谷胱甘肽及相关的调节酶。方法：应该商用试剂盒检测H2O2、MDA含量以及Nrf2下游的抗氧化酶（SOD，CAT）活力、II相代谢酶(GST，NQO1，HO-1)活力、谷胱甘肽（GSH）调节酶类(γ-GCS，GR，GPx)以及GSH和GSSG的含量。结果：铅可以显著提高脂质过氧化物水平，降低抗氧化物酶活力。不同浓度的芒果苷治疗组可以显著降低脂质过氧化物水平，提高抗氧化物酶活力，其中200mg/kg芒果苷的作用最为明显。铅可以显著抑制HO-1、NQO1酶，同时也抑制了GSH相关调节酶，GSH耗竭，GSH/GSSG比例下降。芒果苷治疗组可以提高II相代谢酶以及谷胱甘肽调节酶类，提高GSH含量和GSH/GSSG比例，其中200mg/kg芒果苷的作用最为明显。结论：芒果苷治疗各组可以提高机体氧化还原能力，提高大脑组织及血中Nrf2下游的II相代谢酶以及GSH调节酶类活力，抑制由铅诱导的氧化压力，从而拮抗铅诱导的损伤。以上研究结果提示Nrf2-ARE通路可能参与了芒果苷的氧化应激防御机制。第三部分Nrf2-ARE信号通路在芒果苷拮抗铅致大鼠脑损害中的作用目的：研究Nrf2-ARE通路是否在芒果苷拮抗铅致大鼠脑损害中发挥作用。方法：实时荧光定量PCR (Real-time quantitative Polymerase Chain Reaction，RT-qPCR)、Western Blot，免疫组织化学检测Nrf2、GCLM、GCLC以及HO-1mRNA和蛋白表达；Western-blot检测Nrf2总蛋白和Nrf2核蛋白表达。结果：RT-qPCR表明Nrf2mRNA水平在铅暴露大鼠有较弱的提高，在芒果苷治疗各组也呈较弱的提高。γ-GCS和HO-1在铅暴露大鼠中被抑制，在芒果苷治疗各组中有显著的提高，且呈剂量关系。免疫组化检测显示Nrf2在空白组未阳性神经细胞，在铅暴露大鼠中阳性神经细胞少量表达，在芒果苷治疗各组中表达大幅增加；γ-GCS阳性细胞在铅暴露大鼠中较空白组少，在芒果苷治疗各组中表达大幅增加。结论：Nrf2可被铅激活，芒果苷可以进一步激活它，其调控并非发生于基因转录水平，而可能发生在转录后的Nrf2入核和出核转运水平。Nrf2可能是芒果苷干预铅暴露大鼠抗氧化基因表达的关键转录调控因子。给予芒果苷后Nrf2的激活上调了其下游的γ-GCS、HO-1水平，提示Nrf2-ARE通路可能参与了芒果苷的氧化应激防御机制。综上所述，我们可以得出如下结论：芒果苷能减轻铅暴露大鼠的氧化损伤，对铅暴露大鼠有神经保护作用；这种神经保护作用可能是通过激活Nrf2-ARE通路诱导下游抗氧化/解毒酶等基因的表达从而抑制氧化损伤来实现的；以Nrf2-ARE通路的药物治疗对铅中毒大鼠的防治具有良好的应用前景。Nrf2/ARE信号通路示意图及芒果苷的可能作用机制见图1。本研究的创新之处：将强抗氧化剂芒果苷应用于重金属铅的研究中；从Nrf2通路解释芒果苷对神经系统的保护作用。
[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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114

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