Analysis of signal transduction during ceramide-mediated apoptotic pathway in hypoxic PC12 cell death

缺氧是导致神经细胞死亡的普遍环境刺激之一。在PC12细胞的缺氧培养过程中，我们之前已经证明凋亡细胞的死亡比坏死细胞的死亡更重要，鞘脂，神经酰胺通过激活中性鞘磷脂酶（N-SMase）产生，作为凋亡介质之一。虽然有报道称还原型谷胱甘肽（GSH）似乎可以增强大鼠短暂性前脑缺血后海马神经元的存活，但GSH在缺氧或缺血性损伤中的确切预防机制尚不清楚。我们已经证明GSH通过直接抑制N-SMase活性和神经酰胺形成来保护细胞免受缺氧损伤，从而抑制caspase-3的激活。这些发现表明活性氧（ROS）的产生是缺血或缺氧细胞死亡信号传导的关键事件。因此，下一步研究的目的是了解暴露于H2O2（高活性自由基的重要前体）的PC12细胞凋亡的信号转导途径。令人感兴趣的是，与缺氧细胞死亡途径相比，神经酰胺不参与h2o2诱导的PC12细胞凋亡。这一差异表明PC12细胞在响应氧相关应激时存在多种凋亡途径，如既往研究所示。此外。我们进一步研究了缺氧诱导的PC12细胞死亡期间PLD活性的变化，因为据报道其活性被神经酰胺抑制。随后，PLD2在早期被激活，随后在缺氧细胞死亡通路中下调。此外，在过表达PLD2的PC12细胞中，凋亡细胞数量显著减少。这些结果提高了PLD2激活可能在缺氧诱导的细胞死亡中起抗凋亡作用的可能性。

Hypoxia is one of the pervasive environmental stimuli which lead to neuronal cell death. During hypoxic incubation of PC12 cells, we have previously demonstrated that the apoptotic cell death dominates over the necrotic one and a sphingolipid, ceramide generated through the activation of neutral sphingomyeuinase (N-SMase) functions as one of the apoptotic mediators. Although it has been reported that reduced gluthatione (GSH) appeared to enhance hippocampal neuronal survival after transient forebrain ischemia in rats, the precise preventive mechanism of the action of GSH in hypoxic or ischemic injury remains poorly understood. We have demonstrated that GSH protects cells from hypoxic injury by direct inhibition of N-SMase activity and ceramide formation, resulting in inhibition of caspase-3 activation, These findings suggest that generation of reactive oxygen species (ROS) has been proposed as a crucial event for ischemic or hypoxic cell death signaling. Therefore, the aim of the next study is to understand the signal transduction pathway leading to apoptotic cell death in PC12 cells exposed to H2O2 (an important precursor of highly reactive free radicals). To be interested, compared with hypoxic cell death pathway, ceramide was not involved in H2O2-induced apoptotic cell death of PC12 cells. This difference indicates the existence of multiple apoptotic pathways in PC12 cells in response to oxygen-related stresses as shown in the previous study. Moreover. we further examined the changes of PLD activity during hypoxia-induced PC12 cell death, since its activity is reported to be inhibited by ceramide. Subsequently, PLD2 is activated at the early stage and thereafter down-regulated during hypoxic cell death pathway. In addition, the number of apoptotic cells significantly reduced in PC12 cells overexpressing PLD2. These results raise the possibility that PLD2 activation may play an anti-apoptotic role in hypoxia-induced cell death.