脂氧素A4对小鼠巨噬细胞钙池操纵的钙通道及活性氧的影响

Effects of lipoxin A4 on store-operated calcium channel and production of reactive oxygen species in macrophages

目的研究脂氧素A4对LPS诱导的小鼠RAW 264．7巨噬细胞钙池操纵的钙通道活化及活性氧产生的影响，探讨脂氧素A4保护内毒素性肺损伤的具体机制。方法小鼠RAW 264．7巨噬细胞，随机分为对照组、LPS组、Thapsigargin组、脂氧素A4＋LPS组、脂氧素A4＋Thapsigargin组、2-Aminoethoxydiphenylborate ＋ Thapsigargin组。采用细胞内钙离子特异性荧光探针Flu03／AM和活性氧特异性荧光探针DCFH-DA标记小鼠巨噬细胞。激光共聚焦显微镜动态观察巨噬细胞游离钙浓度变化，流式细胞仪测定活性氧产生变化。结果脂多糖呈剂量依赖升高细胞内游离钙浓度和活性氧的产生。脂氧素A4抑制脂多糖引起的钙内流（抑制率为93％），脂氧素A4同时可抑制Thapsigargin激活钙池操纵的钙通道引起的钙内流（抑制率为75％）。脂多糖诱导巨噬细胞产生大量活性氧（阳性细胞百分比为28．87％±4．5％），脂氧素A4抑制脂多糖诱导巨噬细胞产生活性氧（阳性细胞百分比11．16％±2．5％，P〈0．05）。结论脂多糖通过促进内钙释放而开放钙池操纵的钙通道，大量细胞外钙内流引起钙超载，活性氧大量产生，组织细胞损伤。脂氧素A4可能通过抑制钙池操纵的钙通道，使外钙内流减少，保持细胞钙稳态，减少活性氧的生成而起到抗炎促炎症消退作用。

Objective To investigate the effects of lipoxin A4 on store-operated calcium channel （SOC） and production of reactive oxygen species in macrophages induced by lipopolysaccharide （LPS）. Method Macrophages were randomly assigned to one of the following six groups： control group, LPS group, Thapsigargin group, lipoxin A4 ＋ LPS group, lipoxin A4 ＋ Thapsigargin group, 2-Aminoethoxydiphenylborate ＋ Thapsigargin group. The intracellular [Ca^2＋ ]i was analyzed by confocal laser microscopy. The production of reactive oxygen specips （ROS） was assayed by flow cytometry. Results LPS increased intracellular [Ca^2＋ ] and reactive oxygen species in a dose-dependent manner. Lipoxin A4 suppressed approximately 75 % of the Ca^2＋ entry signal induced by thapsigargin and suppressed approximately 93 % of the Ca^2＋ entry signal induced by LPS. The increase in intracellular [Ca^2＋ ]i was associated with increased ROS production which was abolished in the presence of lipoxin A4. Conclusions These findings indicate that the LPS-induced intracelhilar [Ca^2＋ ]i increase depends on the Ca2~ entry through SOC channel, and lipoxin A4 inhibits Ca^2＋ influx and ROS production through SOC channel in murine macrophages induced by LPS.