【作者】 尹述婷；

【导师】 阮迪云；

【作者基本信息】 中国科学技术大学 ， 神经生物学与生物物理学， 2009， 博士

【摘要】 活性氧介导的氧化损伤参与铅中毒的病理学过程。EGCG是一种高效的自由基清除剂,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。本文以Wistar大鼠和原代培养海马神经元为模型分别研究了EGCG对染铅大鼠海马组织SOD、GSH和MDA含量的影响,铅诱导LTP损伤的影响以及其对铅诱导的海马神经元内活性氧和线粒体膜电位变化的影响。结果显示:EGCG能改善铅暴露Wistar大鼠海马的氧化应激参数;显著增加铅处理的体外培养海马神经元的细胞活力,降低铅处理细胞内的ROS水平,对铅中毒显示一定的防护作用;同时EGCG还能部分地修复铅诱导的Wistar大鼠海马CA1区LTP的损伤。这说明EGCG对铅中毒的保护作用可能与其自由基清除能力有关,且进一步的研究也发现了其对铅诱导的细胞内活性氧升高的抑制作用及对线粒体膜电位的保护作用。因此,EGCG对铅中毒的保护作用可能是通过上述几条途径来实现的。在上述研究中,我们发现,EGCG在高剂量的时候对原代培养海马神经元细胞活力有影响,所以我们进而研究了高剂量时EGCG对细胞活力影响的可能机制。结果显示,EGCG能通过诱导胞外钙离子内流和胞内钙库释放从而增加细胞内游离钙离子浓度,而且这种效应是剂量依赖性的。同时,EGCG还能增加细胞内ROS水平,降低线粒体膜电位,而这些氧化损伤作用可以通过添加维生素E、BAPTA-AM以及EGTA而得到部分修复。另外,高剂量的EGCG还可以降低bcl-2/bax蛋白的表达比率,提高caspase-9/3的酶活,而对乳酸脱氢酶(LDH)泄漏率没有明显影响。这些结果说明,高剂量EGCG对细胞的损伤作用可能是先通过诱导胞内钙超载,然后再通过线粒体途径诱导细胞凋亡的。本文主要是研究EGCG能否改善铅诱导的氧化损伤水平,结果证明了这种假设。因此可以推论EGCG处理的铅应激细胞活力和线粒体膜电位的提高,ROS水平的降低,以及Wistar大鼠海马组织内SOD、GSH活性的增加反映了EGCG对铅诱导的氧化损伤的防护作用,同时这种防护作用也有可能是EGCG对铅暴露大鼠海马CA1区LTP损伤修复的原因之一。本研究的结果也提醒我们,在高剂量的时候,EGCG也具有促氧化作用。因此在应用EGCG作为诊疗制剂或营养保健品时,必须要重视其毒性作用及其使用剂量和使用方式。更多还原

【Abstract】 Recent studies have shown that lead causes oxidative stress by inducing the generation of reactive oxygen species（ROS） and reducing the antioxidant defense system of cells,which suggests that antioxidants may play an important role in the treatment of lead poisoning.As a kind of excellent scavenger of free radicals, modulator of immune systems and chelator of heavy metal,whether Epigallocatechin-3-gallate（EGCG） have the protective effects on altered oxidative stress parameter after lead treatment in vivo and in vitro systems remains unclear.The present study was designed to elucidate whether EGCG have any protective effects on the accumulation of ROS and a decrease of mitochondrial membrane potential（⊿Ψ_m） in lead-exposed cultures of hippocampal neurons and whether EGCG could reverse the changes on redox states in rat hippocampus caused by lead exposure.Our results showed that glutathione（GSH） and superoxide dismutase（SOD） activity decreased accompanied with LTP amplitude decrease in CAl area of hippocampus in the lead-exposed group.EGCG supplementation following lead intoxication resulted in increases in the GSH and SOD levels and increases in the LTP amplitude. Malondialdehyde（MDA） levels,a major lipid peroxidation byproduct,increased following lead exposure and decreased following EGCG treatment.In hippocampal neuron culture model,lead exposure（20μM） significantly inhibited the viability of neurons which was followed by an accumulation of ROS and a decrease of⊿Ψ_m. Treatment by EGCG（10-50μM） effectively increased cell viability,decreased ROS formation and improved⊿Ψ_m in hippocampal neurons exposed to lead.These observations suggest that EGCG is a potential complementary agent in the treatment of chronic lead intoxication through its antioxidative character.Our previous study showed that EGCG has pro-oxidant effects at high concentration.Thus,in this study,we tried to examine the possible pathway of EGCG-induced cell death in cultures of rat hippocampal neurons.Our results showed that EGCG caused a rapid elevation of intracellular free calcium levels([Ca²⁺]_i) in a dose-dependent way.Exposure to EGCG dose- and time-dependently increased the production of ROS and reducedΔΨ_m as well as the Bcl-2/Bax expression ratio. Importantly,BAPTA-AM,EGTA and vitamin E could attenuate EGCG-induced apoptotic responses,including ROS generation,mitochondrial dysfunction,and finally partially prevented EGCG-induced cell death.Furthermore,treatment of hippocampal neurons with EGCG resulted in an elevation of caspase-3 and caspase-9 activities with no significant accompaniment of lactate dehydrogenase（LDH） release, which provided further evidence that apoptosis was the dominant mode of EGCG-induced cell death in cultures of hippocampal neurons.Taken together,these findings indicated that EGCG induced hippocampal neuron death through the mitochondrion-dependent pathway.The present study was designed to elucidate whether EGCG protected altered oxidative stress parameters in Wistar rats and cultures of hippocampal neurons exposed to lead.The hypothesis was evidenced both in EGCG-treated Wistar rats and cell models exposed to lead.Therefore it can be deduced that the increased cell viability andΔψ_m,the decreased ROS accumulation,the increases in the GSH and SOD levels and increases in the LTP amplitude in lead-exposed cultures of hippocampal neurons and lead-exposed Wistar rats,respectively could reflect the antioxidant action of EGCG in lead-treated Wistar rats and neurons.The investigation also placed EGCG at high doses into radical-generating toxicological agents. Therefore,much consideration to safety should be required when EGCG are used as therapeutical reagents or nutrition supplement compounds and the optimum dosage and duration of treatment to obtain better clinical recoveries should also be explored.更多还原