同型半胱氨酸对人离体嗜中性白细胞释放超氧阴离子的影响和机制

Effect and mechanism of homocysteine on stimulus-induced superoxide generation in human neutrophils in vitro

目的用人离体嗜中性白细胞研究同型半胱氨酸(Homocysteine,Hcy)对刺激剂介导的超氧阴离子释放和P47phox磷酸化的影响。方法细胞色素C还原法测定3种刺激剂介导的嗜中性白细胞超氧阴离子释放量。用Westernblot法检测嗜中性白细胞NADPH氧化酶P47phox的磷酸化。结果Hcy呈浓度依赖性促进N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸(N-formyl-methionyl-leucyl-phenylalanine,fMLP)介导的嗜中性白细胞释放超氧阴离子,不影响佛波豆蔻醚乙酸盐(phorbol12-myristate13-acetate,PMA)和花生四烯酸(arachi-donicacid,AA)介导的嗜中性白细胞释放超氧阴离子。酪氨酸激酶抑制剂5,7,45-三羟基异黄酮(genistein)可呈明显抑制fMLP介导的氧自由基产生,而蛋白激酶C抑制剂十字孢碱(staurosporine)仅有轻度抑制作用。Hcy刺激fMLP介导的嗜中性白细胞质因子P47phox磷酸化,与其对超氧阴离子释放的影响是一致的。结论Hcy通过激活膜受体,激活P47phox磷酸化,促进中性白细胞释放超氧阴离子。

[Objective] To study the effect of homocysteine on stimulus-induced superoxide generation and the phosphorylation of P47^phox in huamn neutrophils in vitro. [Methods] The stimulus-induced superoxide generation in human neutrophils was determined using the method of the reduction of cytochrome C. The phosphorylation of P47^phox of NADPH oxidase in neutrophils was detected by Western blotting. [Results] Hcy increased the superoxide generation induced by fMLP in a concentration-dependent manner. Hcy had no obvious effect on the superoxide generation induced by PMA and AA. fMLP-induced superoxide generation was strongly suppressed by an inhibitor of tyrosine kinase, genistein, but was only slightly inhibited by an inhibitor of protein kinase C, staurosporine. In parallel to the effect on the fMLP-induced superoxide generation, Hcy increased fMLP-induced phosphorylation of P47^phox in a concentration-dependent manner. [ Conclusion] Hcy increases the superoxide generation via the activation of membrane receptor-tyrosine kinase pathway and the translocation of P47^phox to cell membrane.