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 - KB 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 sephoridine Pretreatment + LPS group, cells and cell medium were obtained at the 5th, 30ts, 60th, 120th minute after treatment in each group respectively. The expression of mRNA and protein of NF - KB 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 - KB p65 and TNF - αt was not influenced by Sophoridine in RAW264.7 cells. Compared with control group, expression of NF - KB p65 mRNA and protein and TNF - α level was significanfiy increased in LPS group (P ＜0.01 ) 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 - KB p65 mRNA and protein and TNF - α level was significantly deceased in Sophoridine pretreatment + LPS group at same time point(P ＜0.01 ). Conclusion Sophoridine pretreatment can abolish inflammation by LPS through decreasing the expression of NF - KB p65 and TNF - α secretion.
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