槐定碱预处理对LPS诱导RAW264.7细胞表达NF-κB的影响

Effect of Sophoridine Pretreatment on Lipopolysaccharide-induced Expression of NF-κB in RAW264.7 Cells

目的观察槐定碱预处理对LPS诱导RAW264.7细胞NF-κB p65、TNF-α表达的影响,探讨槐定碱抗内毒素机制。方法培养RAW264.7细胞,分为空白对照组、槐定碱预处理对照组、LPS模型组、槐定碱预处理+LPS组。各组处理完毕后5、30、60、120min,分别获取细胞与细胞培养液。Western Blot和RT-PCR分别检测细胞NF-κB p65蛋白与mRNA表达,放免法检测细胞培养液TNF-α含量。结果单独槐定碱对NF-κB p65以及TNF-α表达无影响;LPS模型组各时间点NF-κB p65 mRNA与蛋白以及TNF-α表达均显著高于空白对照组(P<0.01),且随LPS刺激时间延长而持续升高,至120min未见下降;槐定碱预处理+LPS组NF-κB p65mRNA与蛋白及其下游TNF-α表达均较同时间点LPS模型组降低,差异有统计学意义(P<0.01)。结论槐定碱预处理可通过抑制LPS诱导的RAW264.7细胞的NF-κB p65表达及TNF-α分泌发挥抗炎作用。

Objective To investigate the effect and mechanism of Sophoridine pretreatment on express of NF-κB p65 and TNF-α of RAW264.7 macrophages induced by LPS.Methods Cultured RAW264.7 cells were divided them into control group,sophoridine pretreatment group,LPS group and sophoridine Pretreatment ＋ LPS group.cells and cell medium were obtained at the 5th,30th,60th,120th minute after treatment in each group respectively.The expression of mRNA and protein of NF-κB p65 were respectively measured by RT-PCR and Western Blotting.The Cell medium TNF-α level were detected by Radioimmunoassay（RIA）.Results The expression of NF-κB p65 and TNF-α was not influenced by Sophoridine in RAW264.7 cells.Compared with control group,expression of NF-κB p65 mRNA and protein and TNF-α level was significantly increased in LPS group（P0.01） at each time point,and was kept increasing along with LPS stimulation continuing,and no decline phenomenon appeared until the 120th min.However compared with LPS group,NF-κB p65 mRNA and protein and TNF-α level was significantly deceased in Sophoridine pretreatment ＋LPS group at same time point（P0.01）.Conclusion Sophoridine pretreatment can abolish inflammation by LPS through decreasing the expression of NF-κB p65 and TNF-α secretion.