Laboratory of Physiology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece.
PLoS One. 2011;6(10):e25950. doi: 10.1371/journal.pone.0025950. Epub 2011 Oct 18.
Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD) or ischemic preconditioning (IPS). Nerve Growth Factor (NGF) treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N(2)/CO(2) (93:5%) in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.
缺氧是一种严重的应激状态，可诱导细胞死亡，导致发育中和成年期神经系统的神经元丢失。细胞死亡的核心主题是激活不同类别的蛋白酶，如半胱天冬酶、钙蛋白酶和组织蛋白酶。在本研究中，我们研究了这些蛋白酶在缺氧诱导的 PC12 细胞死亡中的作用。大鼠 PC12 是一种与神经系统相关的实验模型细胞系，已经开发了几种用于致死性缺氧（氧和葡萄糖剥夺 OGD）或缺血预处理（IPS）的方案。神经生长因子（NGF）处理的 PC12 分化为交感神经表型，表达轴突和兴奋性。未分化和 NGF 处理的 PC12 细胞在 DMEM 中暴露于 N 2 /CO 2 （93:5%）混合物中，耗尽葡萄糖和丙酮酸钠 16 小时，可建立致死性缺氧。使用蛋白酶特异性抑制剂，如 z-VAD-fmk 用于半胱天冬酶、MDL28170 用于钙蛋白酶和 pepstatin A 用于组织蛋白酶 D 和 E，研究了 caspase、钙蛋白酶和溶酶体组织蛋白酶 D 和 E 对致死性 OGD 诱导的细胞死亡的参与。我们的研究结果表明，pepstatin A 可提供统计学上显著的保护作用，防止未经处理和经 NGF 处理的 PC12 细胞在暴露于致死性 OGD 时发生细胞死亡。我们提出，除了凋亡和坏死这些是致死性 OGD 不可或缺的组成部分的既定过程之外，组织蛋白酶 D 和 E 的激活启动了其他细胞死亡途径，其中这些蛋白酶是关键的参与者。
