氯胺酮抑制人离体中性粒细胞释放超氧阴离子的机制

目的研究氯胺酮的异构体S(+)氯胺酮[S(+)-Ket]和R(-)氯胺酮[R(-)-Ket]对中性粒细胞释放超氧阴离子的影响及机制。方法细胞色素C还原法测定3种刺激剂介导的中性粒细胞超氧阴离子释放量。用荧光分光法检测中性粒细胞内钙的水平。用Western Blot法检测中性粒细胞蛋白质p47phox磷酸化水平。结果S(+)-Ket和R(-)-Ket可抑制N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸(N-formyl-methionyl-leucyl-phenylalanine,fMLP)介导的中性粒细胞释放超氧阴离子(P<0.01)。S(+)-Ket可抑制佛波豆蔻醚乙酸盐(phorbol12-myristate13-acetate,PMA)介导的中性粒细胞释放超氧阴离子和钙离子的增加,而R(-)-Ket可轻度增加PMA介导的中性粒细胞释放超氧阴离子和促进钙离子的增加。S(+)-Ket抑制PMA介导中性粒细胞蛋白质p47phox磷酸化,而R(-)-Ket增强上述作用;钙螯合剂EGTA可取消R(-)-Ket对PMA介导中性粒细胞蛋白质p47phox磷酸化的抑制作用。结论S(+)-Ket通过PKC-钙信息通道,抑制NADPH氧化酶p47phox,抑制超氧阴离子的产生。

Effect of ketamine on stimulus-induced superoxide generation and intracellular calcium in human eutrophils in vitro

Aim To study the effect of two racemic ketamine, S( )-ketamine and R(-)-ketamine on stimulus-induced superoxide generation and intracellular calcium in vitro. Methods The stimulus-induced superoxide generation in human neutrophils was determined by using method of cytochrome C reduction. The intracellular calcium in human neutrophils was measured by chemiluminescence with Fura-2 loading. The phosphorylation of p47phox of NADPH oxidase in neutrophils was detected by Western blotting. Results S( )-Ket and R(-)-Ket inhibited fMLP-induced superoxide generation in neutrophils in a concentration-dependent manner. In the case of PMA, S( )-Ket inhibited PMA-induced superoxide generation and elevation of intracellular calcium of neutrophils in a concentration-dependent manner, whereas R(-)-Ket slightly increased PMA-induced superoxide generation and elevation of intracellular calcium of neutrophils. On the other hand S( )-Ket inhibited the phosphorytion of p47phox of NADPH oxidase subunit,which R(-)-Ket was increased. EGTA can abolished the inhibition of S( )-Ket on PMA-induced phosphorytion of p47phox.Conclusion S( )-Ket inhibits the phosphorylation of p47phox of NADPH oxidase subunit and the superoxide generation in human neutrophils via PKC-calcium signal pathway.