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天然营养素对氧化应激和线粒体功能代谢的调控和作用机制
文献类型：学位论文
| 作者 | 冯智辉 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-10-25 |
| 授予单位 | 中国科学院上海生命科学研究院营养科学研究所 |
| 授予地点 | 中国科学院上海生命科学研究院 |
| 导师 | 陈雁 |
| 关键词 | 二相酶 线粒体功能 谷胱甘肽 |
| 其他题名 | Modulation of oxidative stress and mitochondrial metabolism by natural nutrients and their underlying mechanism |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 第一部分 α-生育酚是一种有效的二相酶诱导剂：对丙烯醛诱导的人视网膜色素上皮细胞的氧化应激和线粒体功能失调的保护作用研究 摘要 维生素E在很久前就被发现是一种脂溶性抗氧化剂。α-生育酚是维生素E的主要成分也是人体内维生素E的主要存在形式。我们推测维生素E除了能够直接和自由基作用发挥抗氧化作用，它还很可能作为二相酶的诱导剂间接地增强细胞的抗氧化防御体系而发挥抗氧化功能。人视网膜色素上皮细胞（ARPE-19）一直被用来研究吸烟和老年性视网膜黄斑病变之间的关系，丙烯醛是吸烟过程中烟雾的主要成份同时也是脂质过氧化的产物，因此我们利用ARPE-19细胞的丙烯醛损伤模型来研究α-生育酚对二相酶的诱导以及对细胞的保护作用。我们发现丙烯醛在75微摩尔的24小时处理的情况下能够显著降低ARPE-19细胞的存活率，抑制Keap1/Nrf2信号通路，降低细胞抗氧化体系，增加细胞氧化损伤，同时损伤线粒体功能。α-生育酚预处理细胞能够激活Keap1/Nrf2信号通路，促进Nrf2由胞浆向核的转运。使一系列二相酶的表达和活性都显著增加，包括谷氨酰胺半胱氨酸连接酶、NAD(P)H苯醌氧化还原酶-1、血红素单加氧酶-1、谷胱甘肽S-转移酶和超氧化物歧化酶，同时总的抗氧化能力和谷胱甘肽的含量也显著增加。抗氧化体系的增强提高了ARPE-19细胞存活率，降低了活性氧自由基和氧化蛋白的水平，改善了线粒体的功能。这些结果都揭示了α-生育酚能够保护丙烯醛引起的细胞损伤，不仅仅是作为一个脂质自由基清除剂，更是作为一个二相酶诱导剂。 第二部分 羟基酪醇在人视网膜色素上皮细胞中激活谷胱甘肽合成从而抑制氧化应激引起的细胞凋亡：Nrf2和JNK-p62/SQSTM1信号通路的激活 摘要 Nrf2-Keap1通路被认为是调控二相酶抵抗氧化应激的关键因素。通过激活Nrf2，细胞保护基因如血红素单加氧酶-1（HO-1）、NAD(P)H苯醌氧化还原酶-1(NQO-1)和谷氨酰胺半胱氨酸连接酶(GCL)被激活。GCL诱导的谷胱甘肽被认为参与细胞氧化还原信号通路、细胞分化和死亡。我们发现叔丁基双氧水（t-BHP）能够引起谷胱甘肽的降低从而诱导人视网膜色素细胞线粒体膜电位的降低和细胞凋亡的发生。羟基酪醇（HT）是橄榄油的一种多酚类化合物。结果显示HT能够激活二相酶增加谷胱甘肽的含量，从而保护线粒体功能预防叔丁基双氧水引起的凋亡。使用谷胱甘肽的特异性清除剂BSO能够明显增加t-BHP造成的损伤，同时还能抑制HT的保护作用。当过表达Nrf2时可以增加谷胱甘肽含量从而抑制t-BHP引起的线粒体膜电位的丢失。同时通过Nrf2的小干扰RNA降低Nrf2表达后，HT诱导的谷胱甘肽的生成和Nrf2靶基因的激活都被显著抑制，这说明HT诱导Nrf2的激是谷胱甘肽的生成的必要条件。另外我们还发现HT能够激活PI3/Akt 和 mTOR/p70S6K信号通路，这两条通路对于细胞存活都有重要的意义，然而当这两条通路被抑制后HT的保护作用依然存在。与他人研究结果不同的是我们发现JNK通路调控的p62/SQSTM1的表达参与到Nrf2的激活。因此我们的研究揭示HT能够通过激活Nrf2来诱导二相酶的活化尤其是谷胱甘肽的生成，从而有效保护氧化损伤引起的线粒体膜电位丢失和细胞凋亡，而HT激活的JNK信号通路很可能参与了这一保护机制的调控。 第三部分 剧烈运动引起的肌肉线粒体功能失调研究：线粒体动态重塑与羟基酪醇的调控作用 摘要 低强度和高强度耐力运动所带来的结果是完全不同的，过度运动（Exe）会引起疲劳和肌肉损伤。然而这其中的分子机制仍不明确，寻找有效药剂来预防疲劳和过度运动引起的损伤是目前体育学研究的重点之一。我们设计大鼠的剧烈运动方案来模拟过度运动从而研究肌肉细胞损伤和线粒体动态重塑在其中的作用。根据实验结果我们发现过度运动容易降低机体的耐受力同时对肾功能和免疫系统都有损害。肌肉萎缩的标志基因Atrogin-1 和MuRF-1的mRNA在过度运动中显著增加，肌肉细胞自吞噬和线粒体裂解也显著增加。过度运动还引起PGC-1α和线粒体复合物I的蛋白表达量下降，激活JNK和Erk1/2信号通路，诱导p53、p21、MnSOD的蛋白表达。我们推测是过度运动引起氧化应激诱导了自吞噬和线粒体功能失调，这一推测在体外肌肉细胞（C2C12）培养中得到证实。羟基酪醇（HT）是天然橄榄油中的一种多酚，它能够有效提高运动耐力同时抑制过度运动引起的肾功能和免疫功能损伤。同时我们还发现过度运动老鼠在摄入HT后自吞噬、线粒体裂解和PGC-1α表达下降都被有效的抑制。另外HT还能有效提高运动大鼠肌肉的线粒体融合，增加线粒体复合物I和II的活性。这些结果揭示过度运动引起的疲劳和肌肉损伤很可能是通过影响线粒体动态重塑来实现的，包括下调线粒体的生成、增加线粒体的裂解和自吞噬的发生。而通过补充HT可以有效调控线粒体动态变化，提高线粒体功能从而有效地预防过度运动导致的肌肉损伤。 |
| 索取号 | D2011-143 |
| 英文摘要 | Part I α-Tocopherol is an effective phase II enzyme inducer: Protective effects on acrolein-induced oxidative stress and mitochondrial dysfunction Abstract Vitamin E has been elucidated as a major lipid-soluble chain-breaking antioxidant in mammals for years. α-Tocopherol is one of vitamin E analogues and the major form in human body. We propose that, besides its direct chain-breaking antioxidant activity, α-tocopherol may exert antioxidant activity through enhancing antioxidant system as a phase II enzyme inducer. In the present study, we investigated the inducing effect on phase II enzymes and the protective effect on acrolein-induced toxicity in human retinal pigment epithelial cell line, ARPE-19. Acrolein is a major component of cigarette smoke and also a product of lipid peroxidation. Acrolein treatment (24 h of 75μmol/L) caused significantly loss of ARPE-19 cell viability, increased oxidative damage, decreased antioxidant defense, and inactivation of Keap1/Nrf2 pathway and mitochondrial dysfunction. This cellular model has been used studying smoking and age-related macular degeneration. Pretreatment with α-tocopherol activated Keap1/Nrf2 pathway by increasing Nrf2 expression and translocation of Nrf2 into the nucleus. Such activity consequently increased the expression and/or activity of phase II enzymes, including glutamate cysteine ligase, NAD(P)H:quinine oxidoreductase 1, heme-oxygenase 1, glutathione S-transferase, and superoxide dismutase. The total antioxidant capacity and glutathione level were also evelated by α-tocopherol treatment. The enhanced antioxidant defense protected ARPE-19 cells from acrolein-induced decrease in cell viability, inhibited reactive oxygen species level and protein oxidation, and improved mitochondrial function. These results suggest that α-tocopherol protects ARPE-19 cell from acrolein-induced cellular toxicity, not only as a chain breaking antioxidant, but also as a phase II enzyme inducer. Part II Prevention of oxidative stress-induced cell apoptosis by hydroxytyrosol in human retinal pigment epithelial cells: Activation of Nrf2 and JNK- p62/SQSTM1 pathways Abstract The Nrf2-Keap1 pathway is believed to be a critical regulator of the phase II defense system against oxidative stress. Cytoprotective genes such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase (NQO-1), and γ-glutamyl-cysteine ligase (GCL) are induced by activation of Nrf2. GCL-induced GSH production is believed to affect redox signaling, cell proliferation and death. Here we report that tert-butyl hydroperoxide (t-BHP)-induced GSH reduction led to mitochondrial membrane potential loss and apoptosis in cultured human retinal pigment epithelial cells from the ARPE-19 cell line. Hydroxytyrosol (HT), a natural phytochemical from olive leaves and oil was found to induce phase II enzymes and GSH, thus protected t-BHP-induced mitochondrial dysfunction and cell apoptosis. Depletion of GSH by buthionine-[S,R]-sulfoximine (BSO) enhanced t-BHP toxicity and abolished HT protection. Overexpression of Nrf2 increased GSH content and efficiently protected t-BHP-induced mitochondrial membrane potential loss. Meanwhile, HT-induced GSH enhancement and induction of Nrf2 target genes (GCLc, GCLm, HO-1, NQO-1) were inhibited by Nrf2 knockdown, suggesting that HT increases GSH through Nrf2 activation. In addition, we found that HT was able to activate the PI3/Akt and mTOR/p70S6-kinase pathways, both of which contribute to survival signaling in stressed cells. However, the effect of HT was not inhibited by PI3K inhibitor LY294002. Rather, c-Jun N-terminal kinase (JNK) activation was found to induce p62/SQSTM1 expression, which is involved in Nrf2 activation. Our study demonstrates that Nrf2 activation induced by the JNK pathway plays an essential role in the mechanism underlying HT’s anti-apoptotic actions through the endogenous antioxidant system. Part III Mitochondrial dynamic remodeling in strenuous exercise-induced muscle and mitochondrial dysfunction: Regulatory effects of hydroxytyrosol Abstract The outcomes of endurance exercise at low intensity vs. high intensity differ and excessive exercise (Exe) at high intensity can cause fatigue and damage to muscle and immune functions. However, the underlying molecular mechanisms are still unclear and effective agents for preventing fatigue and Exe-induced damage are greatly desired. We designed a rodent strenuous exercise protocol to mimic Exe and explored the ensuing cellular damage and involvement of mitochondrial dynamics. We found that Exe was prone to decrease endurance capacity and induce damage to renal function and the immune system. Muscle atrophy markers atrogin-1 and MuRF1 mRNA were increased by Exe, accompanied by increased autophagy and mitochondrial fission in skeletal muscle. Exe caused a decrease in PGC-1α and complex I expression; it also activated JNK and Erk1/2 pathways and consequently induced p53, p21, and MnSOD expression in skeletal muscle. The involvement of oxidant-induced autophagy and mitochondrial dysfunction was confirmed in C2C12 myoblasts. Hydroxytyrosol (HT), a natural olive polyphenol, efficiently enhanced endurance capacity and prevented renal and immune system damage induced by Exe. HT also inhibited the Exe-induced increase in autophagy and mitochondrial fission and the decrease in PGC-1α expression. In addition, HT enhanced mitochondrial fusion and mitochondrial complex I and II activities in muscle of Exe rats. These results demonstrate that Exe-induced fatigue and damage to the functions of muscle and immune systerm may be mediated via the regulation on mitochondrial dynamic remodeling, including downregulation of mitochondrial biogenesis and upregulation of autophagy. HT supplementation may regulate mitochondrial dynamic remodeling and enhance mitochondrial function, and thus improve exercise capacity under Exe conditions. |
| 语种 | 中文 |
| 源URL | [http://202.127.25.144/handle/331004/116]  |
| 专题 | 中国科学院上海生命科学研究院营养科学研究所_信号转导与营养相关疾病研究组 |
| 推荐引用方式 GB/T 7714 | 冯智辉. 天然营养素对氧化应激和线粒体功能代谢的调控和作用机制[D]. 中国科学院上海生命科学研究院. 中国科学院上海生命科学研究院营养科学研究所. 2011. |
入库方式： OAI收割
来源：上海营养与健康研究所
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