富勒醇对大鼠的海马神经元活性以及学习记忆的影响
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摘要
背景：富勒烯及其衍生物由于独特的物理及化学性质,越来越广泛地应用于材料科学、电子科学和纳米技术等领域。富勒醇是富勒烯的羟基化衍生物,是通过化学方法在富勒烯的碳上引入羟基而得到的功能化衍生物。富勒醇保留富勒烯大多数优良性质的同时,多羟基的水溶特性也极大地丰富了其在生物医学领域中的应用。在特定浓度下,富勒醇会保护细胞和组织并体现抗氧化、抗肿瘤和抗病毒等特性,同时在某些浓度时,富勒醇也可能存在潜在毒性导致细胞死亡或细胞凋亡。此外,富勒烯衍生物能有效地穿越脂质膜和具有平衡钙离子水平的能力,因此中枢神经系统可能是一个潜在的富勒烯及其衍生物的作用目标。但是,目前关于富勒醇对神经系统的作用及其可能机制仍然少有关注。本论文旨在研究富勒醇对大鼠海马神经元活性以及学习记忆的影响。
方法：本论文中的研究对象为Wistar大鼠。为了研究富勒醇对神经元活性的影响,本论文在原代培养的海马神经元上采用了MTT细胞增殖与毒性检测、Hoechst33342/PI细胞双染和单细胞凝胶电泳等方法。为了研究富勒醇对大鼠的学习记忆能力的影响,本论文采用了Morris水迷宫行为学分析、在体场电位记录、Golgi银染法和扫描电镜染色等方法。结果：(1)低浓度(25μM以下)的富勒醇提高原代培养海马神经元的细胞活性,高浓度(100μM以上)的富勒醇降低海马神经元的细胞活性；(2)低浓度的富勒醇能提高海马神经元在铅暴露后的细胞存活率,同时,高浓度的富勒醇损伤神经元DNA,导致Caspase蛋白含量升高,并诱导细胞凋亡；(3)低浓度的富勒醇提高海马神经元内的SOD活性和GSH含量,高浓度的富勒醇提高MDA水平,说明富勒醇能够改变海马神经元内的氧化还原水平；(4)发育期给予低剂量富勒醇(腹腔注射5mg/kg)后,大鼠在Morris水迷宫实验中的正确象限停留时间增加和逃避潜伏期缩短；(5)铅暴露会使大鼠在Morris水迷宫实验中的正确象限停留时间减少,富勒醇能修复这种铅暴露引起的学习记忆能力下降；(6)富勒醇不能降低大鼠海马组织中的H2O2水平,也不能提高大鼠海马组织的SOD活性、总抗氧化能力和GSH含量；(7)富勒醇提高大鼠学习记忆能力的过程中能够增加作为突触可塑性重要指标的树突棘数目和阻止铅引起的突触后致密斑数目的减少。
结论和讨论：低浓度富勒醇能修复神经元的氧化损伤,高浓度富勒醇会诱导神经元凋亡,即富勒醇对原代培养的海马神经元具有浓度依赖性作用。由于单体态富勒醇表面携带的羟基能够清除自由基,而聚集态富勒醇的还原能力减弱,所以本研究认为该浓度依赖性作用的机制与其物理特性和神经元内氧化还原水平相关。腹腔注射低剂量富勒醇能提高大鼠学习记忆能力,并部分修复铅暴露引起的学习记忆能力损伤。在富勒醇的作用过程中,富勒醇没有降低大鼠海马中的氧化水平但改变了突触连接的数目,因此本研究认为富勒醇在生物体内的作用可能不依赖于氧化还原水平的变化,而是与神经元突触连接的结构性变化相关。这些都说明富勒醇的生物效应远比我们目前理解的要复杂。本论文为富勒醇在神经系统中的生物学应用和安全性评价提供了理论参考。
BACKGROUND:As a kind of functional water-soluble fullerene derivative, fullerenol has driven extensive applications in biomedical fields. Fullerenol is known to protect cells and tissues as well as to have other properties of antioxidant, antitumor and antivirus at certain lower concentrations, but may have potential toxicity which leads to cell death or apoptosis at higher concentrations. Because fullerene derivatives can effectively get through the lipid membrane and influence calcium homeostasis, the central nervous systems may be a target of fullerene and its derivatives. However, the effect of fullerenol on the nervous systems is not well understood. The present study intended to investigate the effects of fullerenol on hippocampal neuron viability and on learning and mormory in rats.
METHODS:The research was carried out in Wistar rats. The effect of fullerenol on hippocampal neuron viability was investigated with primary cell cultrue, MTT cell proliferation and cytotoxicity assay, Hoechst33342/PI double stain and single cell gel electrophoresis techniques. The influence of fullerenol on learning and memory was studied with in vivo field potential recording, Morris water maze, Golgi staining and scanning electron microscopy.
RESULTS:(1) Fullerenol at low concentrations (below25μM) significantly enhanced neuron viability, and fullerenol at high concentrations (above100μM) reduced neuron viability;(2) Fullerenol at low concentrations improved the survival rate of hippocampal neurons after lead exposure. Fullerenol at high concentrations induced neuronal DNA damage and increased Caspase protein content;(3) Fullerenol could change the level of redox in cultured hippocampal neurons;(4) Fullerenol (5mg/kg intraperitoneal injection) meliorated the ability to locate the hidden platform in water.(5) Fullerenol could repair the damage of learning and memory caused by lead exposure;(6) Fullerenol could not reduce the level of redox in rat hippocampus;(7) Fullerenol significantly increased the number of dendritic spines and prevented reduction in postsynaptic density from lead-exposure, indicating structural changes accompanying the functional improvments and repairements.
CONCLUSION AND DISCUSSION:Fullerenol promotes hippocampal neuron viability and protects these cells from lead-induced damage at lower concetrations, but causes cell death at higher concentrations in rats. The concentration-dependent effects of fullerenol are mainly due to its physical properties and its influence on the reduction-oxidation pathway. Low dose of fullerenol can improve learning and memory and hold a repair capacity of damages caused by lead exposure in Wistar rats. The influences of fullerenol in vivo are due to the functional changes of neuron structure. In fact, the biological function of fullerenol is more complex than we thought. The present study may provide a theoretical basis for biological assessment and for biomedical applications of fullerenol in the neuroscience-related fields.
方法：本论文中的研究对象为Wistar大鼠。为了研究富勒醇对神经元活性的影响,本论文在原代培养的海马神经元上采用了MTT细胞增殖与毒性检测、Hoechst33342/PI细胞双染和单细胞凝胶电泳等方法。为了研究富勒醇对大鼠的学习记忆能力的影响,本论文采用了Morris水迷宫行为学分析、在体场电位记录、Golgi银染法和扫描电镜染色等方法。结果：(1)低浓度(25μM以下)的富勒醇提高原代培养海马神经元的细胞活性,高浓度(100μM以上)的富勒醇降低海马神经元的细胞活性；(2)低浓度的富勒醇能提高海马神经元在铅暴露后的细胞存活率,同时,高浓度的富勒醇损伤神经元DNA,导致Caspase蛋白含量升高,并诱导细胞凋亡；(3)低浓度的富勒醇提高海马神经元内的SOD活性和GSH含量,高浓度的富勒醇提高MDA水平,说明富勒醇能够改变海马神经元内的氧化还原水平；(4)发育期给予低剂量富勒醇(腹腔注射5mg/kg)后,大鼠在Morris水迷宫实验中的正确象限停留时间增加和逃避潜伏期缩短；(5)铅暴露会使大鼠在Morris水迷宫实验中的正确象限停留时间减少,富勒醇能修复这种铅暴露引起的学习记忆能力下降；(6)富勒醇不能降低大鼠海马组织中的H2O2水平,也不能提高大鼠海马组织的SOD活性、总抗氧化能力和GSH含量；(7)富勒醇提高大鼠学习记忆能力的过程中能够增加作为突触可塑性重要指标的树突棘数目和阻止铅引起的突触后致密斑数目的减少。
结论和讨论：低浓度富勒醇能修复神经元的氧化损伤,高浓度富勒醇会诱导神经元凋亡,即富勒醇对原代培养的海马神经元具有浓度依赖性作用。由于单体态富勒醇表面携带的羟基能够清除自由基,而聚集态富勒醇的还原能力减弱,所以本研究认为该浓度依赖性作用的机制与其物理特性和神经元内氧化还原水平相关。腹腔注射低剂量富勒醇能提高大鼠学习记忆能力,并部分修复铅暴露引起的学习记忆能力损伤。在富勒醇的作用过程中,富勒醇没有降低大鼠海马中的氧化水平但改变了突触连接的数目,因此本研究认为富勒醇在生物体内的作用可能不依赖于氧化还原水平的变化,而是与神经元突触连接的结构性变化相关。这些都说明富勒醇的生物效应远比我们目前理解的要复杂。本论文为富勒醇在神经系统中的生物学应用和安全性评价提供了理论参考。
BACKGROUND:As a kind of functional water-soluble fullerene derivative, fullerenol has driven extensive applications in biomedical fields. Fullerenol is known to protect cells and tissues as well as to have other properties of antioxidant, antitumor and antivirus at certain lower concentrations, but may have potential toxicity which leads to cell death or apoptosis at higher concentrations. Because fullerene derivatives can effectively get through the lipid membrane and influence calcium homeostasis, the central nervous systems may be a target of fullerene and its derivatives. However, the effect of fullerenol on the nervous systems is not well understood. The present study intended to investigate the effects of fullerenol on hippocampal neuron viability and on learning and mormory in rats.
METHODS:The research was carried out in Wistar rats. The effect of fullerenol on hippocampal neuron viability was investigated with primary cell cultrue, MTT cell proliferation and cytotoxicity assay, Hoechst33342/PI double stain and single cell gel electrophoresis techniques. The influence of fullerenol on learning and memory was studied with in vivo field potential recording, Morris water maze, Golgi staining and scanning electron microscopy.
RESULTS:(1) Fullerenol at low concentrations (below25μM) significantly enhanced neuron viability, and fullerenol at high concentrations (above100μM) reduced neuron viability;(2) Fullerenol at low concentrations improved the survival rate of hippocampal neurons after lead exposure. Fullerenol at high concentrations induced neuronal DNA damage and increased Caspase protein content;(3) Fullerenol could change the level of redox in cultured hippocampal neurons;(4) Fullerenol (5mg/kg intraperitoneal injection) meliorated the ability to locate the hidden platform in water.(5) Fullerenol could repair the damage of learning and memory caused by lead exposure;(6) Fullerenol could not reduce the level of redox in rat hippocampus;(7) Fullerenol significantly increased the number of dendritic spines and prevented reduction in postsynaptic density from lead-exposure, indicating structural changes accompanying the functional improvments and repairements.
CONCLUSION AND DISCUSSION:Fullerenol promotes hippocampal neuron viability and protects these cells from lead-induced damage at lower concetrations, but causes cell death at higher concentrations in rats. The concentration-dependent effects of fullerenol are mainly due to its physical properties and its influence on the reduction-oxidation pathway. Low dose of fullerenol can improve learning and memory and hold a repair capacity of damages caused by lead exposure in Wistar rats. The influences of fullerenol in vivo are due to the functional changes of neuron structure. In fact, the biological function of fullerenol is more complex than we thought. The present study may provide a theoretical basis for biological assessment and for biomedical applications of fullerenol in the neuroscience-related fields.
引文
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