激动δ-阿片受体对NaN₃诱导原代培养大鼠皮层神经元损伤的保护作用及相关机制的研究

【作者】 朱敏；

【导师】 朱粹青；

【作者基本信息】 复旦大学 ， 神经生物学， 2011， 博士

【摘要】 缺血性脑损伤是一类常见的神经系统疾病,具有发病率高、致残率高及死亡率高的特点,严重危害人类健康。虽然低温、谷氨酸受体拮抗剂等干预措施具备潜在的神经保护作用,但临床上安全有效的治疗措施还未建立。因此,神经元缺血缺氧损伤的保护研究受到了极大关注。阿片受体广泛分布于哺乳动物的中枢神经系统,主要分为δ、μ、κ三个亚型,具有重要的生物学功能。近来研究发现,激动6-阿片受体(DOR)还可以产生抗脑缺血缺氧的保护作用,但DOR的神经保护作用机制至今尚不明晰。线粒体功能损伤是脑缺血缺氧的早期事件,严重的线粒体损伤可以激活线粒体相关凋亡通路,在神经元缺血缺氧损伤中起着非常重要的作用。但目前关于激动δ-阿片受体对神经元线粒体损伤的直接影响尚无研究报道。缺血缺氧损伤可以引起神经元内DOR的表达显著下降,而DOR的表达量是决定缺血缺氧时神经元能否存活的重要因素。虽然通过某些物理方法(如缺血缺氧预适应)或药物处理可以上调DOR的表达,但对其中的机制研究目前还非常匮乏,相关报道提示缺血缺氧损伤可能改变神经元中基因的表观遗传修饰,从而影响DOR的表达。本课题利用线粒体呼吸链复合物IV的抑制剂NaN3处理SD大鼠原代培养皮层神经元建立损伤模型,这是体外模拟缺血性脑卒中的常见模型之一。在该损伤模型上,我们分别观察短时程(30min和60min)和长时程(2天)激动DOR产生的神经保护作用,并探讨各自相关机制。在研究短时程激动DOR的神经保护作用时,我们集中关注：①短时程激动DOR对线粒体功能损伤的影响,包括线粒体膜电势、线粒体内钙浓度的变化、ROS的生成；②短时程激动DOR对线粒体凋亡通路的影响,包括线粒体细胞色素c的释放和caspase-3的激活；③短时程激动DOR对神经元损伤的影响；④介导短时程激动DOR神经保护作用的下游信号转导通路。在探讨长时程激动DOR的神经保护效应时,我们主要研究：①长时程激动DOR对自身受体基因的表观遗传修饰和蛋白表达的影响；②长时程激动DOR对线粒体凋亡通路相关蛋白Bcl-2基因的表观遗传修饰和蛋白表达的影响；③长时程激动DOR对线粒体凋亡通路和神经元损伤的影响；④介导长时程激动DOR神经保护效应的下游信号转导通路。本课题的研究结果如下：第一部分：短时程激动DOR可快速激活PKC/线粒体ERK (mito ERK)通路,逆转NaN3诱导的线粒体功能损伤。激动DOR 30min通过PKC主要激活nito ERK,并通过PKC/mito ERK通路抑制NaN3诱导的神经元线粒体功能损伤,包括线粒体膜电势下降、线粒体钙超载及ROS大量生成；还能通过该通路逆转NaN3引起的线粒体凋亡通路激活,包括线粒体细胞色素c释放和caspase-3活化的增加。激动DOR 60min可通过PKC激活mito ERK和cyto ERK,并通过PKC/ERK通路抑制NaN3诱导的神经元损伤。第二部分：长时程激动DOR可激活PI3K/Akt/NF-κB通路,抑制NaN3诱导的线粒体凋亡通路激活,保护神经元。激动DOR2天主要激活PI3K/Akt/NF-κB通路,并通过该通路抑制NaN3引起的自身受体DOR和线粒体凋亡通路相关蛋白Bcl-2基因启动子组蛋白去乙酰化改变,逆转NaN3诱导的DOR和Bcl-2蛋白表达下调,进而阻止线粒体cyto c的释放和神经元损伤,起到保护作用。综上所述,本课题研究发现,短时程激动DOR通过逆转NaN3诱导的线粒体功能损伤,阻止线粒体凋亡通路的激活；而长时程激动DOR通过影响相关基因的表达,抑制NaN3诱导线粒体凋亡通路的激活。短时程激动DOR和长时程激动DOR的神经保护效应分别由不同的胞内信号转导通路介导。更多还原

【Abstract】 Neuronal death as a result of neuronal injury following hypoxic/ischemic insults, such as stroke, is an irreversible process that leads to long term neurological deficit. The prevention of neuronal injury is therefore critical in rescuing the brain from neurological disaster. However, clinical strategies that may help mitigate the effects of hypoxic/ischemic injury are still very limited.δ-opioid receptor (DOR) is a guanine nucleotide-binding regulatory protein (G protein)-coupled receptor that is widely distributed in different mammalian cells. It is well established in recent years that stimulating delta-opioid receptor (DOR) with its specific agonists elicits neuroprotection against hypoxia/ischemia. However, the underlying mechanisms are not well understood yet.Mitochondria are key players in apoptosis regulation during hypoxia/ischemia. Inadequate oxygen supply perturbs mitochondrial membrane potentials, diminishes their ability to buffer cytosolic calcium, and increases formation of free radicals. Irreversible mitochondrial dysfunction further leads to the opening of mitochondrial permeability transition pore and the release of apoptosis-inducing factors, which constitute a point of no return in cell commitment to death. And suppressing mitochondrial dysfunction helps greatly to prevent neurons from entering mitochondria-dependent cell death pathways during hypoxia/ischemia.DOR is an oxygen-sensitive membrane protein, whose expression is severely suppressed by hypoxia/ischemia. The mechanisms underlying are unclear, though it is suggested that hypoxic/ischemic insults might promote epigenetic reprogramming of DOR expression in neurons.In this investigation, we studied the effects of DOR activation on mitochondrial injury and mitochondria-dependent cell death pathways in rat primary cortical neuronal cultures exposed to sodium azide (NaN3), and explore the possible mechanisms underlying. The exposure of primary cortical neuronal cultures to NaN3 has been widely adopted as a mitochondrial dysfunction model to investigate hypoxia/ischemia-induced injuries and neuroprotection against these injuries in vitro.Here we show that selective DOR activation reverses mitochondrial dysfunction in primary rat cortical neuronal cultures treated with 10 mM NaN3 for 30 min, including mitochondrial membrane depolarization, mitochondrial Ca2+ overload and reactive oxygen species generation. Short-term stimulation of DOR also inhibits cytochrome c release and caspase-3 activation in neurons, and attenuates the severe neuronal injury caused by NaN3 insults. Further study on the mechanisms underlying indicates that DOR activation prevents mitochondrial dysfunction, cytochrome c release and caspase-3 activation induced by NaN3 treatment for 30 min mainly through PKC/mitochondrial ERK pathway.We also find that selectively stimulating DOR attenuates cell damage in primary rat cortical neurons exposed with 80μM NaN3 for 2 days. DOR protein levels in neurons are down-regulated during NaN3 treatment, which is reversed by prolonged DOR activation and may contribute greatly to its neuroprotective effects, since DOR expression is a critical determinant of neuronal survival during prolonged stress. DOR stimulation also reverses the down-regulation of Bcl-2 caused by NaN3 treatment for 2 days, which prevents cytochrome c release and ensuing neuronal death. Further investigation on the mechanisms underlying demonstrates that DOR activation suppresses neuronal injury induced by NaN3 exposure for 2 days mainly through PI3K/Akt/NF-KB pathway. Prolonged DOR stimulation prevents the down-regulation of DOR and Bcl-2 by increasing the binding of NF-κB to their promoters and promoting histone acetylation.更多还原