Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]_i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]_i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]_i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

Costunolide 是一种天然倍半萜内酯，具有多种药理活性，例如神经保护或诱导细胞凋亡和细胞凋亡。然而，木香内酯对人红细胞中促存活因子和酶的影响，例如分别是谷胱甘肽和葡萄糖-6-磷酸脱氢酶 (G6PDH)，尚未得到研究。我们的目标是确定木香内酯诱导的 eryptosis 的潜在机制并逆转这一过程。根据膜联蛋白-V 结合、流式细胞术前向散射的细胞体积和细胞内谷胱甘肽 [GSH] _i估计磷脂酰丝氨酸暴露来自高效液相色谱。通过分光光度法测量细胞内谷胱甘肽的氧化状态和酶活性。用木香内酯处理红细胞剂量依赖性地提高膜联蛋白 V 结合细胞的百分比，减少细胞体积，消耗 [GSH] _i并完全抑制 G6PDH 活性。通过用特定的 PKC-α 抑制剂白屈菜红碱预处理红细胞，木香内酯对膜联蛋白-V 结合和细胞体积的影响显着逆转。然而，后者对木香内酯诱导的 GSH 消耗没有影响。木香内酯诱导细胞凋亡，消耗 [GSH] _i并灭活 G6PDH 活性。此外，我们的研究揭示了白屈菜红碱对木香内酯诱导的 eryptosis 的抑制作用，表明木香内酯和 PKC-α 之间存在关系。此外，白屈菜红碱的作用与 GSH 消耗无关。了解 G6PDH 抑制伴随 GSH 耗竭的机制应该有助于开发抗疟疾治疗策略或基于合成致死率的方法来升级癌细胞中的氧化应激，以使其对化疗和放疗敏感。