Villagrasa V, Cortijo J, Martí-Cabrera M, Ortiz J L, Berto L, Esteras A, Bruseghini L, Morcillo E J
Departamento de Farmacología, Facultad de Medicina y Odontología, Universitat de València, Spain.
J Pharm Pharmacol. 1997 May;49(5):525-9. doi: 10.1111/j.2042-7158.1997.tb06836.x.
It has been suggested that reactive oxygen species released by activated polymorphonuclear leukocytes (PMN) in man is one mechanism of tissue injury. Therapeutic action aimed at increasing antioxidant defence mechanisms is still a clinical challenge. This study examines the activity of N-acetylcysteine, a known antioxidant, in the protection of PMN exposed in-vitro to the chemoattractant peptide fMet-Leu-Phe (FMLP), the protein kinase C activator phorbol myristate acetate or the lipid peroxidation promoter t-butyl hydroperoxide. FMLP (3-300 nM) and phorbol myristate acetate (160 pm-160 nM) induced concentration-related superoxide anion generation. Pre-treatment with N-acetylcysteine (33-333 microM) resulted in concentration-related inhibition of superoxide production induced by FMLP (30 nM) or phorbol myristate acetate (16 nM);-log IC50 values were 3.97 +/- 0.07 and 3.91 +/- 0.10, respectively. Changes in intracellular calcium ion concentration ([Ca2+]i) induced by FMLP (30 nM) were studied in fura-2-loaded human PMN. FMLP produced a transient calcium response, i.e. a peak followed by decay to a residual value above baseline. N-Acetylcysteine (333 microM) did not affect either basal [Ca2+]i values or changes in [Ca2+]i values after treatment with FMLP. Activation by phorbol myristate acetate caused a reduction in glutathione levels from 5.94 +/- 0.86 (control) to 1.84 +/- 0.51 nmol/3 x 10(6) cells (P < 0.05 compared with control). Pre-treatment with N-acetylcysteine (333 microM) fully reversed the reduction in glutathione levels induced by phorbol myristate acetate (4.83 +/- 0.68 nmol/3 x 10(6) cells; P > 0.05 compared with control). Exposure to t-butyl hydroperoxide (0.5 mM, 30 min) markedly increased malondialdehyde levels (from 0.03 +/- 0.02 to 0.73 +/- 0.07 nmol/10(6) cells), and index of lipid peroxidation. Malondialdehyde levels were significantly reduced in PMN treated with N-acetylcysteine (333 microM; 0.55 +/- 0.04 nmol/10(6) cells; P < 0.05 compared with untreated cells exposed to t-butyl hydroperoxide). In conclusion, N-acetylcysteine reduces superoxide generation in response to FMLP and phorbol myristate acetate and partially protects against lipid peroxidation in PMN from man. The protection afforded by N-acetylcysteine is not related to alteration of the intracellular calcium signal but might be effected by replenishment of the intracellular glutathione levels.
有人提出，人类活化的多形核白细胞（PMN）释放的活性氧是组织损伤的一种机制。旨在增强抗氧化防御机制的治疗作用仍是一项临床挑战。本研究检测了已知抗氧化剂N-乙酰半胱氨酸在保护体外暴露于趋化肽fMet-Leu-Phe（FMLP）、蛋白激酶C激活剂佛波酯肉豆蔻酸酯乙酸盐或脂质过氧化促进剂叔丁基过氧化氢的PMN中的活性。FMLP（3 - 300 nM）和佛波酯肉豆蔻酸酯乙酸盐（160 pm - 160 nM）诱导了浓度相关的超氧阴离子生成。用N-乙酰半胱氨酸（33 - 333 microM）预处理导致FMLP（30 nM）或佛波酯肉豆蔻酸酯乙酸盐（16 nM）诱导的超氧产生受到浓度相关的抑制；-log IC50值分别为3.97±0.07和3.91±0.10。在负载fura-2的人PMN中研究了FMLP（30 nM）诱导的细胞内钙离子浓度（[Ca2+]i）变化。FMLP产生了短暂的钙反应，即一个峰值后衰减至高于基线的残余值。N-乙酰半胱氨酸（333 microM）既不影响基础[Ca2+]i值，也不影响用FMLP处理后的[Ca2+]i值变化。佛波酯肉豆蔻酸酯乙酸盐激活导致谷胱甘肽水平从5.94±0.86（对照）降至1.84±0.51 nmol/3×10(6)个细胞（与对照相比，P < 0.05）。用N-乙酰半胱氨酸（333 microM）预处理完全逆转了佛波酯肉豆蔻酸酯乙酸盐诱导的谷胱甘肽水平降低（4.83±0.68 nmol/3×10(6)个细胞；与对照相比，P > 0.05）。暴露于叔丁基过氧化氢（0.5 mM，30分钟）显著增加了丙二醛水平（从0.03±0.02升至0.73±0.07 nmol/10(6)个细胞），即脂质过氧化指标。用N-乙酰半胱氨酸（333 microM）处理的PMN中丙二醛水平显著降低（0.55±0.04 nmol/10(6)个细胞；与暴露于叔丁基过氧化氢的未处理细胞相比，P < 0.05）。总之，N-乙酰半胱氨酸可减少对FMLP和佛波酯肉豆蔻酸酯乙酸盐的反应中超氧生成，并部分保护人类PMN免受脂质过氧化。N-乙酰半胱氨酸提供的保护与细胞内钙信号的改变无关，但可能是通过补充细胞内谷胱甘肽水平来实现的。
