芳香新塔花总黄酮对脂多糖诱导的RAW264.7细胞分泌炎症因子的影响及机制

目的观察芳香新塔花总黄酮(ZCF)对脂多糖(LPS)诱导的小鼠单核/巨噬细胞系RAW264. 7细胞炎症模型中炎症因子的影响及机制。方法采用LPS刺激RAW264. 7细胞,建立细胞炎症模型。以MTT法检测不同浓度芳香新塔花总黄酮对RAW264. 7细胞的毒性作用。芳香新塔花总黄酮和阿托伐他汀钙(阳性药物)预处理,观察各组细胞的形态,以Griess法检测NO的含量,以酶联免疫吸附试验检测细胞上清中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)的含量,实时荧光定量PCR(qRT-PCR)测定TLR4和NF-κB p65 mRNA的表达。结果1μg/m L LPS刺激RAW264. 7细胞24 h,可成功构建炎症细胞模型。芳香新塔花总黄酮在1~100μg/m L浓度范围内对RAW264. 7细胞无显著的细胞毒性。LPS刺激RAW264. 7细胞后,细胞体积明显增大,形态不规则,伸出大量的伪足,而阿托伐他汀钙和芳香新塔花总黄酮干预后,RAW264. 7细胞变小,触角减少,形态较规则。脂多糖可明显诱导RAW264. 7细胞分泌NO、TNF-α、IL-1β和IL-6(P <0. 01),TLR4和NF-κB p65 mRNA表达增高,而芳香新塔花总黄酮和阿托伐他汀钙可使NO、TNF-α、IL-1β和IL-6的释放受到抑制(P <0. 05,P <0. 01)。芳香新塔花总黄酮和阿托伐他汀钙预处理可使TLR4和NF-κB p65 mRNA的表达降低(P <0. 05,P <0. 01)。结论芳香新塔花总黄酮可抑制LPS诱导的RAW264. 7细胞炎症反应,下调NO、TNF-α、IL-1β和IL-6的表达,其机制可能与其抑制TLR4/NF-κB信号转导通路的激活有关。

Effects and Mechanism of Ziziphora clinopodioides Favonoids on Secretion of Inflammatory Factors from Lipopolysaccharide Induced RAW264.7 Cells

Objective To investigate the effects and mechanism of Ziziphora clinopodioides favonoids(ZCF)on the release of inflammatory factors from lipopolysaccharide(LPS)induced RAW264.7 cells.Methods The inflammation model was constructed by treating RAW264.7 cells with lipopolysaccharide(LPS).The toxic effects of different concentrations of ZCF on the RAW264.7 cells were detected by MTT.After pretreatment with ZCF and atorvastatin calcium(positive drug),the cell morphology of each group was observed.The content of NO was detected using Griess and TNF-α,IL-1βand IL-6 were detected by enzyme-linked immunosorbent assay.The mRNA relative expressions of TLR4 and NF-κB p65 were detected by Real-time fluorescence quantitative PCR.Results The model of inflammatory cells was successfully constructed in RAW264.7 cells induced by LPS(1μg/mL)for 24 h.ZCF had no effect on the activity of RAW264.7 in the concentration range of 1-100μg/mL.After LPS stimulating RAW264.7 cells,the cells were significantly larger in size,irregular in shape,and a large number of pseudopodia were outstretched.However,after the intervention of atorvastatin calcium and ZCF,RAW264.7 cells were smaller,less pseudopodia and more regular in shape.The release of NO,TNF-α,IL-1β,IL-6 and the mRNA relative expressions of TLR4 and NF-κB p65 were significantly increased in RAW264.7 cells induced by LPS,ZCF and atorvastatin calcium can inhibit the release of NO,TNF-α,IL-1βand IL-6(P<0.05,P<0.01).Conclusion ZCF can inhibit the inflammatory response in RAW264.7 cells induced by LPS,and down-regulate the expressions of NO,TNF-α,IL-1βand IL-6,which may be related to the inhibition of the activation of TLR4/NF-κB signaling pathway.