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

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.