甘草次酸减轻放射性炎症反应的作用机制研究

Effect of glycyrrhetinic acid attenuating radiation-induced inflammation and its mechanism

目的:探讨甘草次酸(glycyrrhetinic acid,GA)减轻放射性炎症的作用机制。方法:以4Gy X线照射小鼠巨噬细胞RAW264.7为辐射诱导炎症的细胞模型,采用ELISA法检测细胞上清液中的促炎症因子水平,RT-PCR法检测促炎症因子的mRNA表达水平,cell-based ELISA法检测细胞内磷酸化的p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)蛋白表达,凝胶电泳迁移实验(electrophoretic mobility shift assay,EMSA)检测活化蛋白-1(AP-1)的DNA结合活性,荧光酶标仪检测细胞内活性氧(reactive oxygen species,ROS)产生,细胞色素C还原法检测细胞内尼克酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶的活性。结果:甘草次酸通过抑制X线照射后RAW264.7细胞内促炎症因子IL-6、IL-1β的mRNA表达而降低其分泌。甘草次酸抑制p38MAPK磷酸化及下游转录因子AP-1的DNA结合活性。并通过抑制X线照射后RAW264.7细胞内NADPH氧化酶活性而减少ROS的产生。结论:甘草次酸可能通过抑制NADPH氧化酶/ROS/p38MAPK信号通路而抑制电离辐射诱导的炎症反应。

AIM： To investigate the anti-inflammatory effect and underlying molecular mechanisms of glycyrrhetinic acid（ GA） on radiation-induced inflammation. METHODS： 4Gy X-ray irradiated mouse macrophage RAW264. 7 was established as the cell model of radiation-induced inflammation.RAW264. 7 macrophages were pretreated with GA（ 10 ug /ml） before exposure to X-ray irradiation.Protein and mRNA levels of pro-inflammatory cytokines were measured by ELISA and RT-PCR respectively. Phosphorylated p38 mitogen-activated protein kinase（ p38 MAPK） was determined using cell based ELISA. DNA binding activity of activator protein-1（ AP-1） was assessed with nuclear protein extracts by electrophoretic mobility shift assay（ EMSA）. The activity of nicotinamide adenine dinucleotide phosphate（ NADPH） oxidase was determined using cytochrome C reduction assay. RESULTS：18β-GA decreased the secretion of pro-inflammatory cytokines interleukin-（ IL-1β） and IL-6 by reducing mRNA expression levels. After treatment with 18β-GA,p38 MAPK phosphorylation and DNA-binding activity of activator protein-1（ AP-1） were decreased. ROS over-production was alleviated by suppressing NADPH oxidase activity in irradiated RAW264. 7 macrophages. CONCLUSION： GA presents potent anti-inflammatory effects on radiation-induced inflammation by suppressing pro-inflammatory cytokines production through the downregulation of NADPH oxidase / ROS / p38 MAPK signaling pathway in irradiated RAW264. 7 macrophages.