摘要
自由基清除剂——依达拉奉(edaravone)是一种全新的脑细胞保护剂,它通过清除病变部位产生的自由基,阻断神经细胞受损过程,减少受损神经细胞数量,以最大限度保留患者的正常机能。它能够去除脑组织缺氧时出现的过氧化脂质,羟自由基等自由基,从而抑制神经细胞的氧化损伤,实现对神经细胞的保护作用。PrP106-126(氨基酸序列为KTNMKHMAGAAAAGAVVGGLG)是21个氨基酸的多肽,在体外与PrPsc有某些相同的性质,如可形成淀粉样纤维,并具有蛋白酶K抵抗性,其模拟PrPsc的作用已经得到广泛认可。PC12细胞是大鼠肾上腺嗜铬细胞瘤细胞的克隆细胞系,加入神经生长因子(NGF)后发生分化,具有神经元的性质,且易于稳定传代培养,已经广泛应用于神经系统药物干预方面的研究。感染PrP106-126肽段的分化后PC12细胞是一种较为理想的异常朊蛋白毒性作用细胞模型,我们的前期工作已经通过建立异常朊蛋白毒性作用细胞模型,证实氧化应激在朊蛋白病中可能处于始动与核心地位,PrP106-126肽段对分化PC12细胞有毒性作用,使细胞形态学发生改变,使细胞存活率下降,其机制可能与氧化应激作用有关。
本课题通过研究依达拉奉对感染PrP106-126肽段的分化后PC12细胞药物量效与时效的观察,首先确定依达拉奉对细胞的最佳保护作用浓度为60μmol/L。细胞形态学的观察以及流式细胞学的分析表明,依达拉奉能够有效地提高感染PrP106-126肽段的分化后PC12细胞的存活率,抑制细胞的凋亡。本课题研究结果表明:自由基清除剂—依达拉奉对感染PrP106-126肽段的分化后PC12细胞有明显的保护作用。其保护作用是通过抑制感染分化后PC12细胞氧化应激、减轻细胞凋亡来实现的。依达拉奉抑制感染PrP106-126肽段的分化后PC12细胞的凋亡,抗氧化损伤和抗凋亡保护作用的机制可能与caspase依赖的线粒体通路有关:(1)阻止线粒体膜电位的降低;(2)抑制活性氧的产生;(3)降低caspase-3的活性,抑制caspase-3的激活;(3)上调Bcl-2的表达、降低Bax的表达。依达拉奉可能通过清除自由基,保护感染分化后PC12细胞的线粒体功能而发挥其抗氧化损伤、减少细胞凋亡的神经保护作用。同时依达拉奉能够提高细胞内GSH的含量以及SOD的活性,降低MDA的含量,提示依达拉奉亦可能对细胞内源性抗氧化机制起到保护和激活的作用。
朊蛋白病目前尚无有效的治疗方法,本课题通过研究依达拉奉对感染PrP106-126肽段的分化后PC12细胞,即异常朊蛋白毒性作用细胞模型的保护作用,旨在为探索开发治疗朊蛋白病的有效药物提供科学的方法和依据。
Prion protein diseases are infectious, inherited, sporadic spongiform degeneration encephalopathies caused by prion protein conformational change. CJD is the most common form of human being prion protein diseases. CJD is a rare disease that occurs in three forms: sporadic, genetic, and iatrogenic. More recently, a new variants of CJD(nvCJD) has been reported. All forms of transmissible spongiform encephalopathies (TSEs) are characterized by spongiform degeneration of the brain, reactive gliosis, and neuronal loss. They are associated with the accumulation of an abnormal isoform (PrPsc) of the cellular prion protein (PrPc) in the brain. The scrapie prion protein (PrPsc), which is formed from PrPc, is assumed to be the etiological agent of TSE.
PrP106-126 is a synthetic peptide consisting of amino-acid residues 106-126 of human PrPc , is able to polymerize into amyloid-like fibrils in vitro, like PrPsc. It is neurotoxic and induces activation of astroglial and microglial cells in vitro. So PrP106-126 can serve as a model to study the cellular effects of PrPsc.
Edaravone (2-methyl-1-phenyl-2-pyrazolin-5-one, MCI-186) is free-radial scavenger that has been evaluated as a neuroprotective compound which reduces the increase of hydroxyl radical and superoxide anion level in several models of cerebral ischemia. In addition, edaravone not only has antioxident action, but also protects cells from apoptosis. However, little information is available on antioxidant edaravone in regulating oxidative stress and apoptosis such as the Bcl-2/Bax apoptotic pathway via mitochondria after the differentiated PC12 cells infected by prion protein 106-126 peptide at cellular level.
In this experiment, the neuroprotective effects of free scavenger( edaravone ) for the differentiated PC12 cells infected by prion protein 106-126 peptide was investigated. Our results showed that edaravone is a relatively more potent neuroprotective drug for preventing PC12 cells from PrP106-126 damage. First, we found a perfect model to study the cellular toxicity of prion protein 106-126 peptide. Then by observing the nuclear morphology changes and flow cytometric analysis, edaravone showed its significant effect on protecting differentiated PC12 cells against prion protein 106-126 peptide induced apoptotic cell death. In these cells, the level of glutathione(GSH) and activities of superoxide dismutase(SOD) were augmented and malondialdehyde(MDA) were abased. In addition, edaravone also protected against cell damage induced by PrP106-126, which generated hydroxy radicals(.OH) by the Fenton reaction. These results suggest that edaravone is very effective in preventing oxidative stress triggered by deterioration of cellular functions to reduce ROS levels. Edaravone can protect the differentiated PC12 cells infected by prion protein 106-126 peptide against oxidative stress probably due to stimulating endogenous antioxidant defense mechanism.
Treatment with edaravone significantly protected against PrP106- 126-induced apoptosis/necrosis observed the nuclear morphological changes and measured useing flow cytometric analysis. Both necrosis and apoptosis were precisely quantified using propidium ioide(PI) and Annexin-Ⅴdual staining. The apoptosis in PrP106-126-induced PC12 cells was associated with loss of mitochondrial membrane potential, the formation of ROS, GSH depletion, suppressions of SOD, activation of caspase-3 , down-regulation of Bcl-2 and up-regulation of Bax. In contrast, treatment of PC12 cells with edaravone significantly prevented the above-mentioned mitochondrial dysfunction.
In summary, our results extended edaravone neuroprotective mechanism, to determine that edaravone protects PC12 cells from apoptosis through attenuating the damage of mitochondria, suppresses the Bax protein overexpression, elevating the Bcl-2 protein expression on the critical hinge for apoptosis, inhibits the molecular pathway upstream of caspase. Our research showed that edaravone is a very safe and effective neuroprotective drug against oxidative stress, apoptosis and provide a useful therapeutic strategy for the treatment of oxidative stress-induced neurodegenerative disease such as prion protein diseases.
引文
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