Nrf2与MT的协同作用在保护慢性间歇性低氧所致心脏损伤中的作用

发布时间：2018-05-22 10:30

本文选题：呼吸睡眠暂停 + 间歇性低氧　；参考：《吉林大学》2015年博士论文

【摘要】：阻塞性睡眠呼吸暂停（obstructive sleep apnea, OSA）低通气综合征，是一种最常见的严重危害人类健康的睡眠疾病之一。慢性间歇性低氧（chronic intermittent hypoxia, CIH）是OSA的主要病理特征，目前众多学者应用CIH的动物模型研究OSA所致的心脏损伤，发现CIH是引发包括心室重构在内的心脏损伤的主要原因。目前认为，氧化应激是OSA所致心血管疾病的主要发病机制之一。我们在前期研究中发现CIH早期（3天）心肌组织未见明显氧化应激损伤同时伴随金属硫蛋白（metallothionein, MT）表达升高，然而在CIH晚期（4周和8周）MT表达下降、心肌氧化应激水平升高并出现心肌损伤及心功能下降；并且MT心脏特异性过表达转基因（cardiac-specific overexpressing MT gene, MT-TG）小鼠证实MT对CIH所致的心肌氧化应激损伤、心肌纤维化、炎症及心功能不全有明确的保护作用；与此同时MT基因敲除（MT-knockout, MT-KO）小鼠导致CIH诱导的心脏损伤提前出现并且程度加重。NF-E2相关因子2（NF-E2-related factor2,Nrf2）是一种含有亮氨酸拉链基本结构的转录因子，属于Cap-n-colla（rCNC）调节蛋白家族，是机体抵抗内外界氧化和化学等刺激的重要的防御性转导通路，促进细胞内氧化还原环境的调节。目的：明确Nrf2在CIH所致心脏损伤中的作用及其与MT的关系，并探讨Nrf2激动剂莱菔硫烷（Sulforaphane, SFN）保护CIH诱导的心脏损伤的机制。方法：CIH处理方案为20.9%O2/8%O2FIO2交替进行，30次/小时，，12小时/天，最低氧饱和度变化维持在60%-70%，用以模拟发生在中重度OSA患者的氧饱和度状态；正常对照组给予同样周期的间歇性正常气流处理。本实验主要分为三部分：第一部分实验中将Nrf2-TG小鼠和野生型FVB小鼠暴露于CIH4周，Nrf2-KO小鼠和野生型C57BL/6J小鼠暴露于CIH3天和4周，每组小鼠为6只。第二部分中将MT-TG小鼠和野生型FVB小鼠暴露于CIH4周，MT-KO小鼠和野生型129S1小鼠暴露于CIH3天和4周，每组小鼠为6只。为进一步探讨PI3K/Akt/GSK-3β/Fyn通路在调节Nrf2的表达中的作用，将FVB小鼠暴露于CIH3天，同时给予或不给予PI3K抑制剂LY294002，每组小鼠为6只。第三部分实验为研究SFN在CIH诱导的心脏损伤中的保护作用及机制，将MT-KO和Nrf2-KO小鼠及各自的野生型小鼠暴露于CIH4周，同时给予或不给予SFN皮下注射，每组小鼠为6只。结果：CIH早期（3天）小鼠心肌组织中Nrf2表达水平升高、晚期（4周及8周）下降，与MT的表达趋势相同。Nrf2基因心脏过表达小鼠可保护CIH所致的心脏损伤、而Nrf2基因敲除小鼠心脏损伤进一步加重。Nrf2通过与MT的启动子区结合进而促进MT的表达，MT可通过激活PI3K/Akt/GSK-3β/Fyn途径调节Nrf2的功能。SFN可保护MT-KO小鼠中CIH所致的心脏氧化应激损伤和心功能不全，而在Nrf2-KO小鼠中则失去保护作用。结论：在CIH所致的心脏损伤中Nrf2和MT之间存在协同作用关系，二者皆对CIH所致的心脏损伤有保护作用。SFN的保护作用依赖于Nrf2的存在，而不是MT。
[Abstract]:Obstructive sleep apnea obstructive sleep apnea, OSA) hypopnea syndrome (OSAS) is one of the most common sleep diseases that seriously endanger human health. Chronic intermittent hypoxia, CIH) induced by chronic intermittent hypoxia is the main pathological feature of OSA. At present, many researchers use the animal model of CIH to study the heart injury induced by OSA. It is found that CIH is the main cause of cardiac injury, including ventricular remodeling. At present, oxidative stress is one of the main pathogenesis of cardiovascular disease induced by OSA. In our previous study, we found that there was no significant oxidative stress injury associated with increased expression of metallothionein (MTT) in myocardial tissue in the early 3 days of CIH. However, the expression of MT decreased at 4 and 8 weeks in the late stage of CIH. Myocardial oxidative stress level increased, myocardial injury and cardiac function decreased, and MT cardiac-specific overexpressing MT gene, MT-TGG mice confirmed that MT induced myocardial oxidative stress injury and myocardial fibrosis induced by CIH. At the same time, MT-knockout (MT-KOO) mice induced by MT gene knockout (MT-KOO) caused early and aggravated cardiac injury induced by CIH. NF-E2 related factor 2(NF-E2-related factor2Nrf2) is a transcription factor containing leucine zipper basic structure. Cap-n-colla-rCNC regulatory protein family is an important defensive transduction pathway to resist internal and external oxidation and chemical stimulation and promote the regulation of intracellular redox environment. Aim: to investigate the role of Nrf2 in CIH induced cardiac injury and its relationship with MT, and to explore the mechanism of Nrf2 agonist sulforaphane (SFN) in protecting heart injury induced by CIH. Methods 20.9%O2/8%O2FIO2 was performed 30 times / hour for 12 hours / day alternately, and the minimum oxygen saturation remained between 60 and 70 to simulate the state of oxygen saturation in moderate and severe OSA patients. The normal control group was treated with intermittent normal airflow during the same period. The experiment was divided into three parts: in the first part of the experiment, Nrf2-TG mice and wild type FVB mice were exposed to CIH4 week Nrf2-KO mice and wild type C57BL/6J mice to CIH3 days and 4 weeks, 6 mice in each group. In the second part, MT-TG mice and wild-type FVB mice were exposed to CIH4 week MT-KO mice and wild-type 129S1 mice to CIH3 days and 4 weeks. There were 6 mice in each group. In order to further investigate the role of PI3K/Akt/GSK-3 尾 / Fyn pathway in regulating the expression of Nrf2, FVB mice were exposed to CIH3 for days and were given PI3K inhibitor LY294002 at the same time, six mice in each group. In the third part, to study the protective effect and mechanism of SFN on CIH induced heart injury, MT-KO and Nrf2-KO mice and their wild-type mice were exposed to CIH4 for weeks and were given or not injected with SFN subcutaneously. There were 6 mice in each group. Results the expression of Nrf2 in the myocardium of mice was increased in the early 3 days and decreased at 4 and 8 weeks in the late stage. The same trend of the expression of MT. Nrf2 gene can protect the heart injury induced by CIH in the mice with over expression of NRF 2 gene. The cardiac damage in Nrf2 knockout mice was further aggravated. Nrf2 promoted the expression of MT by binding to the promoter region of MT. MT could regulate the function of Nrf2 by activating PI3K/Akt/GSK-3 尾 -Fyn pathway. SFN could protect the heart from oxidative stress induced by CIH in MT-KO mice. Injury and heart failure, In Nrf2-KO mice, however, there was no protective effect. Conclusion: there is a synergistic relationship between Nrf2 and MT in cardiac injury induced by CIH. Both of them have protective effect on heart injury induced by CIH. The protective effect of SFN depends on the existence of Nrf2 rather than MT.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R766

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