豆蔻明对TLR4/MyD88/NF-κB/iNOS信号通路的调节作用

The regulatory effect of cardamonin on TLR4/MyD88/NF-κB/iNOS pathway

目的探讨豆蔻明(cardamonin,CDN)对RAW264.7小鼠巨噬细胞Toll样受体4(toll-like receptor 4,TLR4)/My D88/NF-κB/i NOS信号通路的调节作用。方法利用脂多糖(lipopolysaccharide,LPS)处理RAW264.7细胞建立炎性细胞模型并分组:正常对照组(Vehicle组)、模型组(LPS组)和药物处理组(LPS+CDN组);CCK-8方法检测细胞活力,Griess法检测细胞培养上清一氧化氮(nitric oxide,NO)含量,RT-PCR检测诱导型NO合成酶(inducible nitric oxide synthase,i NOS)、环氧化酶-2(cyclooxygenase-2,COX-2)、单核细胞趋化蛋白-1(monocyte chemotactic protein 1,MCP-1)、肿瘤坏死因子(tumor necrosis factor,TNF)-ɑ、白介素(interleukin,IL)-1β和IL-6的mRNA表达,Western blot检测i NOS、TLR4、髓样分化因子88(myeloid differentiation factor 88,My D88)、核因子-κB(nuclear factorκB,NF-κB)phosphorylated(p)-p65、inhibitorκBα(IκBα)和p-IκBα的蛋白表达。结果1~50μmol·L^(-1)豆蔻明对RAW264.7细胞没有毒性,但可以剂量依赖性抑制LPS诱导的NO分泌和i NOS、COX-2、MCP-1、TNF-α、IL-1β及IL-6的mRNA表达,25μmol·L-1豆蔻明可下调LPS诱导的i NOS、TLR4、My D88、p-NF-κB p65和p-IκBα蛋白表达及抑制IκBα降解。结论豆蔻明通过抑制TLR4/My D88/NF-κB/i NOS信号通路从而抑制NO的产生。

Aim To assess the regulatory effects of cardamonin( CDN) on toll-like receptor( TLR)-4 /My D88 / NF-κB / i NOS signaling pathway in lipopolysaccharide( LPS)-stimulated RAW264. 7 macrophage cells. Methods LPS-stimulated RAW264. 7 cells were divided into three groups: vehicle-treated group,LPS-treated group and LPS + CDN-treated group. Cell viability was assessed by CCK-8 assay. The concentration of nitric oxide( NO) in cell culture medium was measured by Griess reagent. The mRNA levels of i NOS,COX-2,MCP-1,TNF-α,IL-6 and IL-1β were determined by reverse transcription real-time quantitative PCR( RT-q PCR). The protein levels of inducible nitric oxide synthase( i NOS),TLR4,myeloid differentiation factor 88( My D88),nuclear factor κB( NF-κB) phosphorylated( p)-p65,inhibitor κBα( IκBα),and pIκBα were determined by Western blot. Results 1 ~50 μmol·L^(-1)CDN had no cytotoxicity in RAW264. 7cells. However,CDN inhibited the LPS-induced secretion of nitric oxide( NO) and mRNA expressions of i NOS,COX-2,MCP-1,TNF-α,IL-6 and IL-1β in a dose-dependent manner. Moreover,50 μmol · L^(-1)CDN inhibited the LPS-induced up-regulation of i NOS,TLR4,My D88,NF-κB p-p65,p-IκBα and down-regulation of IκBα. Conclusion Cardamonin inhibits the production of NO via a mechanism associated with the inhibition of TLR4 / My D88 / NF-κB / i NOS pathway.