线粒体复合体Ⅲ抑制剂抗霉素A对巨噬细胞免疫功能的影响

Effect of mitochondrial complexⅢ inhibitor antimycin A on immune function of macrophages

目的研究线粒体复合体Ⅲ抑制剂抗霉素A(AMA)对巨噬细胞免疫功能的影响,并探讨其可能的作用机制。方法 AMA 0.0005,0.05,0.5,5和10 mg·L-1与RAW264.7巨噬细胞分别作用2,24和48 h后,WST-1法检测巨噬细胞增殖;AMA 0.0005,0.05和0.5 mg·L-1与RAW264.7作用2 h后,定量荧光法检测巨噬细胞产生活性氧水平、线粒体膜电位和对白色念珠菌的吞噬作用;Western蛋白印迹法检测丝裂原活化蛋白激酶(MAPK)P38蛋白磷酸化水平;Griess法检测细胞培养上清一氧化氮含量。结果 AMA作用2 h对RAW264.7巨噬细胞增殖无明显影响,当作用时间延长到24和48 h后,可显著抑制RAW264.7巨噬细胞增殖(P<0.01)。AMA作用2 h可显著诱导RAW264.7巨噬细胞产生活性氧(P<0.01),降低线粒体膜电位(P<0.01)。AMA可增强RAW264.7巨噬细胞对白色念珠菌的吞噬功能,显著降低脂多糖诱导的巨噬细胞炎症介质一氧化氮的产生(P<0.01)。AMA显著诱导MAPK P38磷酸化(P<0.01)。结论 AMA能够诱导RAW264.7巨噬细胞线粒体损伤,增强巨噬细胞对白色念珠菌的吞噬功用,降低脂多糖诱导的炎症反应,可能与激活MAPK P38磷酸化,进而激活MAPK信号转导通路有关。

OBJECTIVE To investigate the role of macrophage mitochondrial complex Ⅲ inhibitor antimycin A （AMA） on the immune function of macrophages and to explore its possible mechanism. METHODS AMA of different concentrations （0.0005-0.5 mg·L^-1） was used for incubation with macro- phages RAW264.7 for 2, 24 and 48 h. Cell proliferation was detected by WST-1 assay. Reactive oxygen species （ROS） production, mitochondrial membrane potential and phagocytosis of C.a/bicans were detected by fluorometric assay. Phosphorylation of mitogen-activated protein kinases （MAPK） P38 was detected by Western blotting. Griess reagent was used to determine nitric oxide （NO） production. RESULTS AMA 0.0005-50 mg· L^-1 treatment for 2 h had no obvious effect on macrophages RAW264.7 proliferation, but for 24 and 48 h,the cell proliferation was significantly inhibited （P〈0.01）. AMA 0.005-0.5 mg·L^-1 for 2 h significantly induced ROS production （P〈0.01） and reduced the mitochondrial membrane potential of RAW264.7（P〈0.01 ）, suggesting that the macrophage mitochondrial were damaged. When macrophages＇ mitochondrial complex Ⅲ was impaired, phagocytosis of C.albicans by macrophage was significantly increased （P〈0.01）. Moreover, AMA significantly reduced NO production after the macrophages were stimulated by LPS. AMA 0.05-0.5 mg·L^-1 strongly activated the phosphorylation of MAPK P38 （P〈0.01）. CONCLUSION AMA can impair the mitochondrial function of macrophages, enhance the phagocytic efficiency of C.albicans, and show anti-inflammatory effect. The possible mechanism is to activate the MAPK signal cascade by stimulating the phosphorylation of MAPK P38.