血人参中分离的异甘草素对巨噬细胞的影响及其抗炎作用机制研究

Effect of isoliquiritigenin isolated from blood ginseng on macrophages and its anti-inflammatory mechanism

目的研究血人参中异甘草素的抗炎作用及其机制。方法采用四唑盐比色法(MTT assay,MTT)检测异甘草素对小鼠巨噬细胞的细胞毒;利用脂多糖(lipopolysaccharide,LPS)诱导巨噬细胞和中性粒细胞活化模型,给予无细胞毒剂量的异甘草素处理细胞;通过格里斯法(Griess)检测异甘草素对巨噬细胞释放一氧化氮(nitric oxides,NO)的影响;实时荧光定量聚合酶链反应(real-time fluorescence quantitative PCR,qRT-PCR)检测异甘草素对巨噬细胞炎症因子mRNA转录的影响;酶联免疫吸附法(enzyme linked immuno sorbed assay,ELISA)检测异甘草素对巨噬细胞分泌炎症因子的影响;利用流式细胞术检测异甘草素对中性粒细胞活化和释放活性氧的影响;蛋白质印迹法(western blot,WB)检测异甘草素对巨噬细胞中受体激活蛋白(receptor activator of NF-κB,NF-κB),丝裂素活化蛋白激酶(mitin-activated protein kinase,MAPKs)、干扰素调节因子3(interferon regulator factor 3,IRF3)和信号传导及转录激活蛋白家族(signaling and transcription activator proteins family,STATs)信号通路活化的影响。结果异甘草素在无细胞毒剂量下能显著抑制巨噬细胞释放NO,能抑制肿瘤坏死因子(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1β,IL-1β)、巨噬细胞炎症蛋白-1α(macrophage inflammatory protein-1,MIP-1α)、单核细胞趋化因子-1(monocyte chemotactic protein-1,MCP-1)和干扰素-β(interferon-β,IFN-β)等炎症因子的mRNA转录,同时抑制炎症因子TNF-α、IL-6、MCP-1和IFN-β的分泌;另外对中性粒细胞释放活性氧也具有显著抑制作用;Western blot结果显示异甘草素能够抑制应激活化蛋白激酶1/2(stress-activated protein kinase 1/2,JNK1/2)、细胞外调节蛋白激酶(extracellular regulated protein kinase1/2,ERK1/2)和信号转导和转录活化因子1(signaling and transcription activator proteins 1,STAT1)的磷酸化。结论血人参中异甘草素能够通过抑制JNK1/2、ERK1/2和STAT1信号通路抑制巨噬细胞和中性粒细胞的功能,从而发挥抗炎作用。

Objective The study aims to reveal the anti-inflammatory effects and mechanisms of isoliquiritigenin,a natural compound isolated from the roots of Indigofera stachyodes.Methods Cytotoxicity of ISL on macrophages was detected by MTT method.The effect of isoliquiritigenin on NO release in LPS-induced macrophages was analyzed with griess reagent.qRT-PCR was used to detect the transcription of inflammatory cytokines in macrophages.The secretion of inflammatory cytokines by macrophages was detected by ELISA.Flow cytometry was used to analyze the effect of isoliquiritigenin on the activation of neutrophils.The NF-κB,MAPKs,IRF3,and STATs signaling pathways were detected by Western Blot.Results Isoliquiritigenin significantly inhibited the release of NO from macrophages at the doses without cytotoxicity.Furthermore,isoliquiritigenin inhibited the levels of TNF-α,IL-6,IL-1β,MIP-1α,MCP-1,and IFN-β,and suppressed the secretion of TNF-α,IL-6,MCP-1,and IFN-β.Isoliquiritigenin also reduced reactive oxygen species production of neutrophils.The results of Western Blot showed that isoliquiritigenin inhibited the phosphorylation of JNK1/2,ERK1/2,and STAT1.Conclusion Isoliquiritigenin can inhibit the activation of macrophages and neutrophils through suppressing JNK1/2,ERK1/2,and STAT1 signaling pathways.