槐定碱与TLR4/MD-2阻断剂对LPS诱导的RAW264.7巨噬细胞TLR4-NF-κB-TNF-α通路的影响

Effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264.7 macrophage TLR4-NF-κB-TNF-α

目的:研究槐定碱及TLR4/MD-2阻断剂对LPS诱导的RAW264.7巨噬细胞TLR4-NF-κB-TNF-α通路的影响,探讨其抗内毒素的药理机制。方法:培养RAW264.7巨噬细胞,分为5组:空白对照组,LPS模型组,槐定碱+LPS组,TLR4/MD-2阻断剂+LPS组,TLR4/MD-2阻断剂+槐定碱+LPS组,各组分别于处理完毕后120 min时收集细胞及细胞培养液。West-ern blot检测TLR4蛋白表达,免疫细胞化学检测NF-κB蛋白的分布与表达,逆转录聚合酶链反应(RT-PCR)检测NF-κB,TNF-αmRNA表达,放射免疫分析法检测细胞培养液中TNF-α含量。结果:与LPS模型组比较,槐定碱+LPS组TLR4蛋白,NF-κBmRNA与NF-κB入核率,TNF-αmRNA及培养液中TNF-α含量均显著降低(P<0.01);TLR4/MD-2阻断剂+LPS组与TLR4/MD-2阻断剂+槐定碱+LPS组NF-κB mRNA及NF-κB入核率,TNF-αmRNA及培养液中TNF-α含量均低于LPS模型组(P<0.01),接近空白对照组,但2组之间上述各值的差异均无统计学意义。结论:TLR4/MD-2可能是槐定碱作用的靶位之一,槐定碱抑制TLR4-NF-κB-TNF-α通路活化可能是其抗内毒素机制之一。

Objective： To study the effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264. 7 macrophage TLR4-NF-KB-TNF-α in and its pharmacological mechanism of antiendotoxin. Method ： RAW264. 7 macropha- ges were cultured and divided into 5 groups, namely the blank control group, the LPS model group, the sophoridine ＋ LPS group, the TLR4/MD-2 blocking agent ＋ LPS group and the anti-TLR4/MD-2 ＋ sophoridine ＋ LPS group. Cells and cell culture fluids were collect- ed at 120 min after the each group was processed. The expression of TLR4 protein was measured by western blot, the distribution and ex- pression of NF-KB protein were measured by immunocytochemistry, and the expression of NF-KB and TNF-α mRNA were measured by western blot and reverse transcriptase polymerase chain reaction （RT-PCR）. The content of TNF-α in the cell supernatant was detected by using radioimmunoassay. Result： Compared with the LPS group, the expression of TLR4 protein, NF-KB mRNA, the rate of NF-KB entry the nucleus, TNF-α mRNA and TNF-α content in the cell supernatant were significantly decreased in the sophoridine ＋ LPS group （P 〈0. 01 ）. The rate of NF-KB entry the nucleus and TNF-α in the TLR4/MD-2 blocking agent ＋ LPS group and the TLR4/MD-2 bloc- king agent ＋ sophoridine ＋ LPS group were notablly lower than that of the LPS model group （P 〈 0. 01 ）, close to that of the blank control group. However, there was no statistical significance between the two groups. Conclusion： TLRd/MD-2 may be one of sophoridine＇s tar- gets. Sophordine＇s inhibitory effect on pathway activity of TLR4-NF-KB-TNF-α may be one of its antiendotoxin mechanisms.