【作者】 李好文；

【导师】 杨克敌； 邓家刚；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2013， 博士

【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。更多还原

【Abstract】 Mangiferin(MGN) is an effective antioxidant, and it was reported to chelate withmetals and relieve toxicity caused by heavy metals such as mercury, cadmium and arsenic.These properties make it possible to relieve the toxicity caused by lead. Lead is aubiquitous environmental and industrial pollutant. Exposure to excessive amounts of lead isespecially harmful to the central nervous systems of infants and young children, andoxidative stress has been reported as a major mechanism of lead-induced toxicity.Recent studies have shown that Nrf2-ARE pathway has neuroprotective effect. In thecentral nervous system, Nrf2plays an important role by protecting neurons againstoxidative stress. Nrf2promotes the transcription of a series of genes such asGlutathione-S-transferase (GST)，glutamate cysteine ligase(GCLC and GCLM)，NAD(P)H:quinone oxidoreductase1(NQO1), heme oxygenase-1(HO-1).In our study, molecular biological technique were used to evaluate①the influence ofMGN on the structure and function of nervous system; the influence of MGN on leadburden.②The influence of MGN on lipid peroxidation and the Nrf2downstream enzymesin the cerebral cortex and serum.③the role of Nrf2-ARE pathway in the protective effectof MGN in lead-exposed rats.Major findings are as follows:Part I The influence of MGN on the structure, function of nervous systemand the influence on the lead burden.Objective: To evaluate the influence of MGN on the structure, function of nervoussystem and the influence on the lead burden.Methods:96weaned Wistar rats were divided into six groups (n=16in each group,half male and half female): five groups exposed to500ppm of lead acetate in the drinkingwater and one group as blank control. After8weeks, MGN (50,100,200mg/kg bodyweight) and DMSA were orally administrated to intoxicated groups for four supplementalweeks; the other intoxicated group was left as lead-exposed model group. After the four weeks of administration, all rats received Morris water maze training and test. Rat brainsections were detected by HE staining and transmission electron microscopy(TEM). Blood,femur, brain, liver and kidne lead were determined by ICP-MS.Results: During the course of the Morris water maze experiment, compared with blankcontrol group, the lead-exposed group showed no significant difference. While comparedwith the lead-exposed group, the MGN-treated group(200mg/kg) showed significantdifferences in spatial probe test(p<0.05). Under TEM, in the region CA1of lead-exposedgroup, the changes include neuropile vacuole, abnormal dense bodies in cytoplasm andlysosome in the peripheral vessels, pyknotic compact of gliocyte, etc. While in theMGN-treated groups, the anomaly was mild. The cells in the CA1area in hippocampuswere almost normal.Compared with the lead-exposed group, MGN-treated group loweredthe lead concentration in blood, bone and brain significantly (p<0.05), the effect of MGN inbone and brain showed no significant difference between DMSA group and MGN-treatedgroup(200mg/kg).Conclusion: In our study, compared with blank control group, the lead-exposed groupshowed no deficit in spatial learning.It may be due to the neural compensation. MGN canimprove the spatial learning in the MGN-treated group, compared with the lead-exposedgroup. MGN may ameliorate histopathological lesion in hippocampus. MGN can chelatewith lead in vivo, that maybe due to its chelating property.Part II The influence of mangiferin on lipid peroxidation and the Nrf2downstream enzymes in the cerebral cortex and serum.Objective: To study if MGN intervene the lipid peroxidation and the Nrf2downstreamenzymes.Methods: Commercial kit were used to detect H2O2，MDA，and the Nrf2downstreamantioxidant enzymes, phase II detoxification enzymes and GSH related enzymes andGSH/GSSG content.Results: Lead can significantly improve the level of lipid peroxides, reduce the antioxidantenzyme activity.MGN-treated groups could significantly reduce the level of lipidperoxidation, improve the activity of antioxidant enzymes, phase II detoxification enzymes and GSH related enzymes, wherein the200mg/kg mangiferin has the most obviouseffect.Conclusion: The results of the above studies showed that MGN-treated groups canimprove the activity of antioxidant enzymes, phase II detoxification enzymes and GSHrelated enzymes, inhibit the oxidative stress induced by lead, and thus resist thelead-induced damage. It indicated that Nrf2-ARE maybe involved in the antioxidantmechanism of MGN.Part III The role of Nrf2-ARE pathway in protective effects of MGN inlead-exposed ratsObjective: To study the role of Nrf2-ARE pathway in protective effects of MGN inlead-exposed rats.Methods: Real-time quantitative polymerase chain reaction(RT-qPCR),Western-blotand immunohistochemistry detection were used to detect the expression of Nrf2, GCLC，GCLM，HO-1mRNA and protein expression；Western-blot was used to detect theexpression of nuclear Nrf2protein and total Nrf2.Results: RT-qPCR showed that Nrf2mRNA levels in lead-exposed group andMGN-treated groups increased. γ-GCS， HO-1mRNAand protein expression wereinhibited in lead-exposed group, while in MGN-treated group, they were improvedsignificantly and were in a dose-dependent manner.In immunohistochemistry results, noNrf2positive cells in blank control group were observed. A few in lead-exposed groupwere observed, while lots of Nrf2positive cells in MGN-treated group were observed.γ-GCS positive cells in lead-exposed group were less than that in blank control group, theamount in MGN-treated groups increased significantly.Conclusion: Nrf2can be activated by lead, while MGN can further activate it，theregulation is not at the level of gene transcription，but may be in the post-transcriptionallevel.Nrf2may be the critical transcription factor when MGN intervene the expression ofantioxidant genes. γ-GCS、HO-1levels were raised after the activation of Nrf2, it indicatedthat Nrf2-ARE pathway take part in protective effects of MGN in lead-exposed rats. In summary, we can draw the following conclusions: MGN can relieve the oxidativestress caused by lead, it has neuroprotective effects to lead-exposed rats. The effects wereachieved by the activation of the Nrf2downstream genes such as antioxidant enzymes,phase II detoxification enzymes and GSH related enzymes.The innovation of this study: Antioxidant mangiferin was used in the lead(Pb) study;Nrf2pathway was used to explain the neuroprotective effect of MGN.更多还原