【作者】 王笑亮；

【导师】 王汉东；

【作者基本信息】 南京大学 ， 外科学， 2011， 博士

【摘要】 第一章绪论本章旨在介绍本论文相关研究背景,包括脊髓损伤；Keap1-Nrf2-ARE的结构及激活模式；Nrf2的神经保护作用；Nrf2与NF-κB之间的相互关系以及莱菔硫烷的药代动力学。第二章Nrf2-ARE通路激活对大鼠脊髓损伤的神经保护作用脊髓损伤(spinal cord injury, SCI)继发性损伤常导致氧化应激损伤、炎症反应、线粒体功能丧失的产生,最终导致细胞死亡。核因子E2相关因子2-抗氧化反应元件(nuclear factor E2-related factor 2 antioxidant response element, Nrf2-ARE)通路能有效地调节上述继发性损伤机制。因此,在本研究中我们探讨Nrf2-ARE通路激活是否对大鼠脊髓损伤具有神经保护作用。使用NYU冲击仪制得雌性Fischer大鼠胸段(T8)脊髓中度损伤模型。损伤后,Nrf2蛋白在脊髓组织多种神经细胞(神经元、星形细胞、小胶质细胞和少突胶质细胞)细胞质内活化、积聚并逐渐向细胞核内转移,并启动ARE调控的保护因子如血红素加氧酶1(heme oxygenasel, HO-1)和谷氨酸半胱氨酸链接酶催化亚单位(glutamate-cysteine ligase catalytic subunit, GCLC)的表达。莱菔硫烷(Sulforaphane, SFN)作为Nrf2-ARE通路激动剂能有效地提高大鼠脊髓组织中Nrf2及GCLC的含量,同时抑制核因子κB (nuclear factor-KB, NF-κB)的活化以及降低炎症因子如白介素1β(interleukin-1β,IL-1β)和肿瘤坏死因子α(tumor necrosis factor-α, TNF-α)的表达,最终减少脊髓损伤体积并改善大鼠运动协调性。这些结果表明,Nrf2-ARE的激活对大鼠脊髓损伤具有神经保护作用,同时SFN也可作为治疗脊髓损伤的神经保护药物。Ⅲ第三章SFN抑制ATP诱导的NF-κB激活的作用第二章实验研究已经证实,莱菔硫烷(sulforaphane, SFN)可以有效地提高大鼠损伤脊髓组织中核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)及下游保护因子的表达,同时抑制核因子κB (nuclear factor-κB, NF-κB)的激活及下游炎症因子的表达,最终改善大鼠运动功能。然而,Nrf2与NF-κB之间具体的作用机制尚未明确。本章节实验旨在使用原代大鼠神经元培养,明确Nrf2的抗炎作用及Nrf2与NF-κB具体相互作用机制。原代大鼠神经元使用不同浓度SFN (0, 10μM和20μM)预处理2小时,随后加入1mM ATP诱导NF-κB通路的激活。使用蛋白免疫印迹法检测Nrf2及下游因子、炎症因子和phospho-IκBα的含量。结果显示,SFN呈浓度依赖性的提高大鼠神经元中Nrf2及下游保护因子的含量,同时抑制细胞中ATP诱导的Caspase-1、白介素1β(Inteluekin-1β, IL-1β)及肿瘤坏死因子α(tumor necrosis factorα, TNF-α)的表达；另外,IKBα的磷酸化程度及IL-1β前体(pro-IL-1β)表达也被明显抑制,提示ATP诱导的NF-κB通路被SFN抑制。综上所述,SFN不仅能上调大鼠神经元内Nrf2及下游保护因子的表达,同时抑制ATP诱导的NF-κB活化及下游因子表达。本实验揭示了Nrf2与NF-κB之间复杂的相互作用中一种新的作用机制。第四章Nrf2-ARE在神经元、星形细胞和小胶质细胞损伤后的表达在第二章中,激光共聚焦方法已经证实在大鼠脊髓损伤1天后,核因子E2相关因子2(nuclear factor E2-related factor 2, Nrf2)能在脊髓前角区域内的神经元、星形细胞、少突胶质细胞以及小胶质细胞中激活。但是,Nrf2-ARE在各种细胞损伤后的表达情况目前还没有研究。在本章中,使用可控性的细胞损伤仪对各种神经细胞进行损伤,然后使用蛋白免疫印迹法测定Nrf2及下游因子在细胞损伤后的蛋白水平,明确Nrf2-ARE通路在各种神经细胞损伤后的表达情况。在神经元中,Nrf2-ARE通路在损伤后15分钟即被激活。在星形细胞中,Nrf2蛋白水平在损伤后随时间推移而逐渐降低,下游因子蛋白水平也未见升高；在小胶质细胞中,Nrf2激活在细胞损伤后1小时出现,晚于在神经元中的激活。这些结果表明,Nrf2-ARE通路在各种神经细胞损伤后的表达各不相同,提示各种神经细胞在组织中的功能及作用各不相同。各种细胞类型中Nrf2-ARE具体的神经保护机制以及各种细胞之间的相互作用值得进一步研究。更多还原

【Abstract】 ChapterⅠIntroductionIn this chapter, the background of this study is introduced, including the reviews of spinal cord injury, structure and activation of Keap1-Nrf2-ARE pathway, neuroprotection of Nrf2, interactions between Nrf2 and NF-κB and pharmacokinetic of sulforaphane. Chapter II Activation of the Nrf2-ARE Pathway is Neuroprotective Following Spinal Cord InjuryThe activation of oxidative damage, neuroinflammation and mitochondrial dysfunction has been implicated in secondary pathomechanisms following spinal cord injury (SCI). These pathophysiological processes lead to cell death and are tightly regulated by nuclear factor E2 related factor 2 antioxidant response element (Nrf2-ARE) signaling. Here, we investigated whether activation of Nrf2-ARE is neuroprotective following SCI. Female Fischer rats were subjected to mild thoracic SCI (T8) using the New York University injury device. As early as 30 min after SCI, levels of Nrf2 transcription factor were increased in both nuclear and cytoplasmic fractions and remained elevated for 3 days. Treatment of injured rats with sulforaphane (SFN), an activator of Nrf2-ARE signaling, significantly increased levels of Nrf2 and glutamate-cysteine ligase (GCL), a rate-limiting enzyme for synthesis of glutathione, and decreased levels of inflammatory cytokines, interleukin-1β(IL-1β) and tumor necrosis factor-a (TNF-α) thus leading to a reduction in contusion volume and improvement in coordination. These results show that activation of the Nrf2-ARE pathway following SCI is neuroprotective and that SFN presents a viable compound for neurotherapeutic intevention in blocking pathomechanisms following SCI. ChapterⅢSulforaphane suppresses ATP-induced nuclear factor-κB activation via Nrf2 in primary rat neuronsPrevious studies have shown treatment with sulforaphane (SFN), an activator of nuclear factor E2-related factor 2 (Nrf2), significantly increased levels of Nrf2 and downstream signalings, and decreased levels of inflammatory cytokines by suppressing nuclear factor-KB (NF-κB) pathway. However, the precise mechanism is not fully documented. The role of this study was to investigate the molecular mechanisms of Nrf2 involved in anti-inflammation by depressing NF-κB in primary rat neurons. Dosage of 1 mM ATP was added for 45 min to induce NF-κB activation. Cells were pretreated without SFN, or with SFN (10μM, 20μM) for 2 hrs. Immunoblotting was performed to detect levels of Nrf2 and downstream signalings, cytokines, and phospho-1κBα. SFN increased levels of Nrf2 and downstream signalings, and attenuated the ATP-induced increase of Caspase-1, IL-1βand TNF-α, in a concentration-dependent manner. Consequently, phosphorylation of IκBαand synthesis of pro-IL-1βwere inhibited, which indicated NF-κB pathway was depressed. This study suggests one possible molecular mechanism of Nrf2 anti-inflammation ability by regulating NF-κB. Chapter IVExpression of Nrf2-ARE in neurons, astrocytes and microglia after injury:an in vitro studyIn Chapter II, it is demonstrated by using laser scanning confocal scanning, that Nrf2 is activated in motor neurons, astrocytes, oligodendrocytes and microglia in the ventral horn of cords 1 day following spinal cord injury (SCI) in rats. However, Nrf2 expression pattern of each cell type after injury remains unknown. In this chapter, a controlled cell injury model was employed to investigate Nrf2 expression in rat neurons, astrocytes and microglia. Immunoblotting was carried out to determine levels of Nrf2 protein and downstream proteins. Nrf2-ARE was activated 15 min after injury in neurons; In astrocytes, trauma decreased Nrf2 levels time-dependently, no significant changes were observed in HO-1 and NQ01; Nrf2 activation is delayed than in neurons following injury. This study described the Nrf2 expression in each cell type after injury, however further studies are needed to accomplish the precise mechanisms of Nrf2 neuroprotection in each cell type and the interactions between cell types after injury.更多还原