脂氧素A_4对脂多糖诱导小胶质细胞内活性氧自由基生成的机制研究  

Study on mechanism of lipoxin A_4 reducing reactive oxygen species generation in microglial cell induced by lipopolysaccharide

作　　者：翟恒[1] 吴艳[1] 王芳[1] 孙圣刚[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院神经内科,武汉430022

出　　处：《临床神经病学杂志》2016年第1期38-41,共4页Journal of Clinical Neurology

摘　　要：目的探讨脂氧素A_4(LXA_4)对脂多糖(LPS)诱导的小鼠小胶质细胞株BV2细胞内活性氧自由基(ROS)生成的影响及其机制。方法以100 ng/ml LPS刺激体外培养的BV2细胞为炎症模型。LPS组用含0.035%乙醇的无血清培养基处理30 min后,LXA_4+LPS组用含LXA_4(100 nmol)的无血清培养基预处理30 min后,各亚组在相应时间点分别加入LPS(100 ng/ml)。用荧光酶标仪分别检测各组ROS的荧光值。免疫印迹法检测细胞浆与细胞膜上p47phox蛋白的表达,免疫荧光共聚焦显微镜观察BV2细胞p47phox亚基的膜移位。结果与空白对照组比较,LPS组10 min、20 min、30 min、60 min、6 h、12 h和24 h ROS水平均显著升高(均P<0.05)。与LPS组相应时间点相比,LXA_4+LPS组10 min、20 min、30 min、60min、6 h、12 h和24 h ROS水平均显著降低(均P<0.05)。与LPS组比较,空白对照组、LXA_4组及LXA_4+LPS组胞浆p47phox蛋白表达水平均显著增高(均P<0.05)。与LPS组比较,空白对照组、LXA_4组及LXA_4+LPS组胞膜p47phox蛋白表达水平均显著降低(均P<0.05)。与空白对照组比较,LPS组BV2胞浆p47phox荧光值减少;与LPS组比较,LXA_4组及LXA_4+LPS组胞浆p47phox荧光值增多。结论 LXA_4可以抑制LPS诱导的BV2细胞内烟酰胺腺嘌呤二核苷酸氧化酶亚基p47phox的膜移位及ROS的生成,对小胶质细胞发挥了抗炎症与促炎症消退的保护作用。Objective To investigate the effect and mechanism of lipoxin A4 （ LXA4 ） reducing reactive oxygen species （ROS） generation in microglial cell-BV2 ceils induced by lipopolysaccharide （LPS）. Methods Inflammatory model of BV2 cells was 100 ng/ml LPS culture in vitro. BV2 cells in LPS group was cultured in 0. 035% alcohol serum-free medium by 30 min, and BV2 cells in LXA4 ＋ LPS group was cultured in LXA4 （ 100 nmol） serum-free medium 30 min. Then LPS （ 100 ng/ml） was added to each subgroup at the time point. ROS fluorescence was measured by fluorescence microplate reader. The expression of p47phox protein in cell membrane and cytosolic were measured by by Western blotting, and detection of membrane location of p47phox of BV2 cell was subunited with co-immunofluorescence focus microscope. Results Compared with the control group, ROS levels at 10 min, 20 min, 30 min, 60 min, 6 h, 12 h, 24 h in LPS group were significantly increased （all P〈0.05）. Compared with LPS group at the corresponding time point, ROS levels at 10 min, 20 min, 30 min, 60 min, 6 h, 12 h, 24 h in LXA4 ＋ LPS group were significantly lower （ all P 〈 0.05）. Compared with LPS group, p47phox protein expression in cytosolic in the control group, LXA4 group and LXA4 ＋ LPS group were significantly increased （ all P 〈 0.05）. Compared with LPS group, p47phox protein expression in membrane in the control group, LXA4 group and LXA4 ＋ LPS group were significantly decreased （ all P 〈 0. 05 ）. Compared with the control group, fluorescence value of cytosolic p47phox was reduced in LPS group. Compared with LPS group, fluorescence value of cytosolic p47phox was increased in LXA4 group and LXA4 ＋ LPS group. Conclusion LXA4 can suppress the production of ROS in the LPS-induced of BV2 cells, inhibition of triphosphopyridine nucleotide oxidase subunit p47phox membrane translocation, thus play a protective role of anti-inflammatory and pro-inflammatory effect in microglia.

关 键 词：脂氧素A4 脂多糖 活性氧自由基 烟酰胺腺嘌呤二核苷酸氧化酶 神经炎症 

分 类 号：R741[医药卫生—神经病学与精神病学]