晚期氧化蛋白产物对人巨噬细胞产生NO、ROS的影响及复方黄甘提取物的干预  

Effect of advanced oxidation protein products on NO and ROS in macrophages and role of Compound Huanggan Extract intervention

作　　者：毛萍[1] 莫立乾[1] 肖小燕[1] 宋少练 杨西晓[1] 

机构地区：[1]南方医科大学南方医院药学部,广东广州510515

出　　处：《中成药》2013年第8期1587-1593,共7页Chinese Traditional Patent Medicine

基　　金：广东省科技计划新药开发重点项目资助(2011A030100007);广东省科技计划项目(2012B031800128)

摘　　要：目的研究晚期氧化蛋白产物对人巨噬细胞产生一氧化氮(NO)和活性氧(ROS)的影响以及复方黄甘提取物(大黄、甘草等)对其的干预。方法人巨噬细胞与晚期氧化蛋白产物共同培养24 h,再给予复方黄甘提取物继续培养24 h后,用Griess还原法测定培养上清一氧化氮(NO)的水平变化,RT-PCR法测定诱导型一氧化氮合酶(iN-OS)的基因表达变化,DCFH-DA荧光探针法测定ROS水平的变化;分别用NF-κB抑制剂吡咯烷二硫代氨基甲酸盐、NADPH氧化酶抑制剂二亚苯基碘和复方黄甘提取物预处理细胞1 h,再用晚期氧化蛋白产物刺激,24 h培养结束后测定ROS水平的变化,RT-PCR和ELISA法分别测定细胞因子TNF-α、IL-1β和IL-6的基因表达及蛋白分泌。结果晚期氧化蛋白产物丧失了诱导人巨噬细胞产生NO的能力,但能升高ROS的产生量,同时TNF-α、IL-1β和IL-6的基因表达和蛋白分泌也显著性升高。当给予复方黄甘提取物继续培养后,可显著性地增加NO水平,降低ROS水平;当给予复方黄甘提取物预处理细胞时,也能降低ROS水平,同时降低TNF-α、IL-1β和IL-6的基因表达和蛋白分泌。结论复方黄甘提取物延缓肾衰的进程可能是通过产生NO来减轻其微炎症反应和通过消除ROS来减弱其氧化应激损伤途径实现的。AIM To investigate the effect of advanced oxidation protein products on nitric oxide（NO） and reactive oxygen（ROS） in macrophages and modulated by Compound Huanggan Extract（Rhei Radix et Rhizoma,Glycyrrhizae Radix et Rhizoma,etc.）.METHODS THP-1 macrophages were incubated with advanced oxidation protein products for 24 h and then with various concentrations of Compound Huanggan Extract for another 24 h.NO production in the supernatants of the culture media was determined spectrophotometrically using Griess method,the gene expression of iNOS was detected by RT-PCR and ROS production was evaluated by measuring the fluorescent product from the oxidation of an oxidant-sensitive 2,7-dichlorefluorescin（DCFH）.Meanwhile,THP-1 macrophages were pre-incubated with NADPH oxidase inhibitor,diphenyleneiodonium and nuclear factor kappa-B（NF-κB） inhibitor,pyrrolidine dithiocarbamate or Compound Huanggan Extract prior to incubation with advanced oxidation protein products.Then ROS production was measured.The gene expressions and protein secretions of TNF-α,IL-1β and IL-6 were tested respectively by real time quantitative PCR and ELISA.RESULTS Advanced oxidation protein products had lost the ability of inducing NO production.Nevertheless,it could raise ROS production and the gene expressions and protein secretions of TNF-α,IL-1β,IL-6.While subsequently stimulated by Compound Huanggan Extract,NO production increased significantly.However,ROS production and the gene expressions and protein secretions of TNF-α,IL-1β,IL-6 were dramatically suppressed.CONCLUSION The experiment shows that Compound Huanggan Extract is effective in delaying renal failure in that it could alleviate inflammatory response by inducing NO and lessen oxidative stress injury by eliminating ROS.

关 键 词：晚期氧化蛋白产物 巨噬细胞 一氧化氮 活性氧 

分 类 号：R285.5[医药卫生—中药学]