Watson R W, Rotstein O D, Nathens A B, Dackiw A P, Marshall J C
Department of Surgery, Toronto Hospital, University of Toronto, Canada.
Surgery. 1996 Aug;120(2):150-7; discussion 157-8. doi: 10.1016/s0039-6060(96)80282-0.
Intracellular glutathione, an endogenous antioxidant, protects cellular function against oxidative stress. Because oxidative stress has been implicated in neutrophil apoptosis, we hypothesized that reduced thiol levels may induce apoptosis through an alteration in cellular redox state.
Human polymorphonuclear leukocytes (PMNs), were incubated with medium or with increasing concentrations of the reduced glutathione (GSH)-depleting agents diethylmaleate and diamide and buthionine sulfoximine, an inhibitor of GSH synthesis. Apoptosis was assessed by means of flow cytometry with propidium iodide DNA staining and confirmed morphologically. GSH was measured colorimetrically, and tyrosine phosphorylation was assessed by means of immunoblotting.
Diethylmaleate and diamide induced a dose-dependent reduction in GSH and a corresponding increase in PMN apoptosis. This effect could be reversed with N-acetylcysteine, suggesting that diethylmaleate induces apoptosis through the depletion of GSH. The antioxidant pyrolidine dithiocarbamate had no effect. Because oxidants can mediate intracellular signaling via tyrosine phosphorylation, we therefore evaluated the effects of the tyrosine kinase inhibition on diethylmaleate-induced PMN apoptosis. Both genistein and herbimycin A reduced diethylmaleate-induced apoptosis and tyrosine phosphorylation.
Sulfhydryl oxidation by diethylmaleate alone induces apoptosis, providing evidence of a redox-sensitive, thiol-mediated pathway of apoptosis. Furthermore, tyrosine phosphorylation appears to play an important role in this process. Because apoptosis is a critical mechanism regulating PMN survival in vivo, manipulation of PMN intracellular thiols may represents a novel therapeutic target for the regulation of cellular function.
细胞内谷胱甘肽作为一种内源性抗氧化剂，可保护细胞功能免受氧化应激的影响。由于氧化应激与中性粒细胞凋亡有关，我们推测巯基水平降低可能通过改变细胞氧化还原状态诱导凋亡。
将人多形核白细胞（PMN）与培养基或与浓度递增的谷胱甘肽（GSH）消耗剂马来酸二乙酯、二酰胺以及谷胱甘肽合成抑制剂丁硫氨酸亚砜胺一起孵育。通过碘化丙啶DNA染色利用流式细胞术评估凋亡，并进行形态学确认。采用比色法测定谷胱甘肽，并通过免疫印迹法评估酪氨酸磷酸化。
马来酸二乙酯和二酰胺诱导谷胱甘肽呈剂量依赖性降低以及PMN凋亡相应增加。这种效应可被N - 乙酰半胱氨酸逆转，提示马来酸二乙酯通过消耗谷胱甘肽诱导凋亡。抗氧化剂吡咯烷二硫代氨基甲酸盐无作用。由于氧化剂可通过酪氨酸磷酸化介导细胞内信号传导，因此我们评估了酪氨酸激酶抑制对马来酸二乙酯诱导的PMN凋亡的影响。染料木黄酮和赫曲霉素A均减少了马来酸二乙酯诱导的凋亡和酪氨酸磷酸化。
仅马来酸二乙酯引起的巯基氧化即可诱导凋亡，这为凋亡的氧化还原敏感、巯基介导途径提供了证据。此外，酪氨酸磷酸化似乎在此过程中起重要作用。由于凋亡是体内调节PMN存活的关键机制，操纵PMN细胞内巯基可能代表调节细胞功能的一种新的治疗靶点。
