桉柠蒎油抑制脂多糖诱导的RAW264.7细胞炎症因子产生的作用及其机制研究

Study on the Inhibitory Effect of Eucalyptol,Limonene and Pinene Oil Treatment on the Production of Inflammatory Mediators in Lipopolysaccharide-induced RAW264.7 Cells and Its Mechanism

目的探究桉柠蒎油(ELP)抑制脂多糖(LPS)刺激的RAW264.7细胞炎症因子产生的作用及作用机制。方法构建LPS刺激的RAW264.7炎症细胞模型。采用噻唑蓝(MTT)法检测细胞活性;采用Griess法检测一氧化氮(NO)的产生;采用酶联免疫吸附法(ELISA)测定LPS处理后培养液中炎症因子的浓度;采用Western-blot法检测蛋白表达水平;采用免疫荧光技术检测LPS处理后核转录因子κB亚基(p65)、激活子蛋白1亚基(c-Jun)和干扰素调节因子3(IRF3)的入核情况。结果ELP在6.25~400μg/mL的剂量范围内对LPS处理的RAW264.7细胞活力没有明显抑制作用。ELP(50~300μg/mL)能有效地抑制LPS诱导的RAW264.7细胞NO、前列腺素E2(PGE2)、肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、巨噬细胞炎性蛋白1α(MIP-1α)、单核细胞趋化蛋白1(MCP-1)、白介素-1β(IL-1β)和受激活调节正常T细胞表达和分泌因子(Rantes)的产生。ELP能剂量依赖性地降低Toll样受体4(TLR4)信号通路关键蛋白kappa B抑制因子激酶α/β(IKKα/β)、TANK结合激酶1(TBK1)、干扰素调节因子3(IRF3)、细胞外调节蛋白激酶(ERK1/2)、p38、p65、核因子kappa B抑制蛋白α(IκBα)、c-Jun和c-Jun氨基末端激酶(JNK)的磷酸化水平。ELP同时能抑制LPS诱导的RAW264.7细胞p65、c-Jun和IRF3的入核。结论ELP能剂量依赖性地抑制LPS诱导的RAW264.7细胞炎症因子的产生,其作用机制与阻断TLR4/NF-κB、TLR4/AP-1和TLR4/IRF3信号通路有关。

Objective To explore the effect of eucalyptol,limonene and pinene oil(ELP)on the production of inflammatory mediators in lipopolysaccharide-stimulated RAW264.7 macrophages and its underlying mechanism.Methods The lipopolysaccharide(LPS)-induced RAW264.7 cells were established as the inflammatory cell model.Cell viability was determined by MTT assay.The release of nitric oxide(NO)was measured by Griess assay.The content of inflammatory cytokines and chemokines in culture medium was detected by enzyme linked immunosorbent assay(ELISA).Protein expression was determined by Western-blotting.The nuclear translocation of p65,c-Jun and IRF3 was detected by immunofluorescence assay.Results ELP treatment at concentrations of 6.25-400μg/mL had no inhibitory effect on the cell viability.The production of inflammatory mediators,including NO,prostaglandin E2(PGE2),tumour necrosis factorα(TNF-α),interleukin-6(IL-6),macrophage inflammatory protein 1α(MIP-1α),monocyte chemotactic protein 1(MCP-1),interleukin-1β(IL-1β)and regulated upon activation,normal T cell expressed and secreted(Rantes)was effectively inhibited by ELP(50-300μg/mL)in LPS-induced RAW264.7 cells.ELP treatment concentration-dependently suppressed the expression levels of key components involved in Toll-like receptor 4(TLR4)signaling pathway,such as phosphorylated IKKα/β,TBK1,IRF3,ERK1/2,p38,p65,IκBα,c-Jun and JNK in LPS-stimulated RAW264.7 cells.Moreover,ELP treatment suppressed the nuclear translocation of p65,c-Jun and IRF3 in LPS-induced RAW264.7 cells.Conclusion ELP treatment concentration-dependently suppressed the production of inflammatory mediators in LPS-induced RAW264.7 cells,the underlying mechanism of this action is related to its suppressing effect on the TLR4/NF-κB,TLR4/AP-1 and TLR4/IRF3 signaling pathways.