异黄柏酮酸对LPS诱导RAW264.7细胞炎症的抑制作用  

Inhibitory effects of isoobacunoic acid on LPS-induced inflammation in RAW264.7 cells

作　　者：何冰心 尚海[2] 李凌宇[2] 李靖荣 邹忠梅[2] 宛蕾 HE Bing-xin;SHANG Hai;LI Ling-yu;LI Jing-rong;ZOU Zhong-mei;WAN Lei(School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,China;The Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100193,China)

机构地区：[1]贵州医科大学基础医学院,贵州贵阳550025 [2]中国医学科学院北京协和医学院药用植物研究所,北京100193

出　　处：《中成药》2023年第5期1469-1475,共7页Chinese Traditional Patent Medicine

基　　金：中国医学科学院医学与健康科技创新工程项目(2022-I2M-1-017);国家“重大新药创制”科技重大专项(2019ZX09735002)。

摘　　要：目的探讨异黄柏酮酸对脂多糖(LPS)诱导小鼠单核巨噬细胞白血病细胞(RAW264.7)炎症的作用及机制。方法采用CCK-8法检测异黄柏酮酸对RAW264.7细胞活性的影响,Griess法及荧光法测定一氧化氮(NO)水平,酶联免疫吸附试验(ELISA)检测细胞上清中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、前列腺素E 2(PGE 2)和单核细胞趋化蛋白-1(MCP-1)水平,免疫荧光法检测NF-κB p65核转位,蛋白质免疫印迹(Western blot)法检测诱导型一氧化氮合酶(iNOS)、环氧化酶-2(COX-2)和NF-κB、MAPK信号通路相关蛋白表达。结果异黄柏酮酸能降低LPS刺激RAW264.7细胞释放的NO水平(P<0.05),其IC_(50)为(39.6±3.05)μmol/L;同时,异黄柏酮酸降低了炎性介质PGE 2、TNF-α、IL-6、IL-1β和MCP-1水平(P<0.05),且在0~200μmol/L浓度条件下对RAW264.7细胞活性无明显影响(P>0.05)。进一步研究表明,异黄柏酮酸可降低iNOS、COX-2蛋白表达,抑制IκBα蛋白降解和p65蛋白核转位,下调p38和ERK磷酸化蛋白表达(P<0.05)。结论异黄柏酮酸具有一定的抗炎作用,其机制与抑制MAPK/NF-κB信号通路有关。AIM To investigate the effects and mechanism of isoobacunoic acid on lipopolysaccharide(LPS)-induced inflammation in RAW264.7 cells derived from mouse mononuclear macrophage leukemia cell line.METHODS The RAW264.7 cells had changes in their isoobacunoic acid-induced viability detected by the CCK-8 method;their nitric oxide(NO)level determined by Griess method and fluorescence method;their levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),prostaglandin E 2(PGE 2)and monocyte chemoattractant protein-1(MCP-1)in the cell supernatant detected by enzyme-linked immunosorbent assay(ELISA);their NF-κB p65 nuclear translocation detected by immunofluorescence assay;and the expressions of inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2),NF-κB and MAPK signaling pathway-related proteins detected by Western blot.RESULTS With an IC_(50)value range of(39.6±3.05)μmol/L,isoobacunoic acid reduced the level of NO released by LPS-stimulated RAW264.7 cells(P<0.05).At the same time,it decreased the levels of proinflammatory mediators PGE 2,TNF-α,IL-6,IL-1βand MCP-1(P<0.05),and it left no significant impact on RAW264.7 cells viability at concentrations of 0-200μmol/L(P>0.05).Further studies revealed that the isoobacunoic acid reduced the expression of iNOS and COX-2 proteins,inhibited the degradation of IκBαprotein and nuclear translocation of p65 protein,and down-regulated the expressions of p38 and ERK phosphorylated proteins(P<0.05).CONCLUSION Isoobacunoic acid may find its anti-inflammatory working mechanism lying in the inhibition of MAPK/NF-κB signaling pathway.

关 键 词：异黄柏酮酸 炎症 脂多糖 RAW264.7 NF-κB MAPK 

分 类 号：R285.5[医药卫生—中药学]