幽门螺杆菌VacA通过激活NF-κB诱导巨噬细胞分泌及凋亡

Helicobacter pylori VacA up-regulates secretion of macrophages by activating nuclear factor κB

目的研究幽门螺杆菌（Helicobacter pylori）空泡毒素（VacA）单一毒力决定簇对THP-1巨噬细胞分泌和凋亡的影响，以及核因子κB（nuclear factor κB，NF—κB）在调节VacA诱导的THP-1巨噬细胞功能中的作用。方法将pDsRed—Monomer—C1／vacA转染THP-1巨噬细胞，ELISA法检测巨噬细胞培养上清IL-1β、TNF-α含量；Griess试剂分析培养上清的一氧化氮（NO）水平；荧光探针DCFH-DA检测培养上清的活性氧（ROS）；流式细胞仪检测细胞的凋亡率；凝胶阻滞试验（electrophoretic mobility gel shift assay，EMSA）检测NF—κB的核转位。结果重组质粒转染后6h，重组质粒组培养上清中TNF-α、IL-1β含量明显高于阴性对照组（P〈0．05）；且分别于转染后6h或24h到达峰值；转染后6h或12b，重组质粒组培养上清中NO、ROS含量明显高于阴性对照组（P〈0．05），且于转染后24h达到高峰。转染后16h，重组质粒组的凋亡率明显升高，与阴性对照组相比，差异有统计学意义（P〈0．05）。重组质粒组加NF—κB的特异性抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）后，IL-1β、TNF-α、NO、ROS的分泌量和细胞的凋亡率明显降低，与重组质粒组相比差异有统计学意义（P〈0．05）。EMSA试验显示，转染后3h细胞核内有活化的NF—κB，且于转染后12h活性最强。结论VacA蛋白瞬时高表达上调THP-1巨噬细胞分泌IL-1β、TNF-α、NO和ROS；VacA蛋白瞬时高表达诱导THP-1巨噬细胞凋亡；NF—κB可能参与调节VacA诱导的THP-1巨噬细胞的分泌和凋亡。

Objective To study the effect of vacA on the secretion of THP-1 macrophages as an individual virulence determinant, and the effect of NF-κB on the secretion of THP-1 macrophages. Methods The recombinant plasmid pDsRed-Monomer-C1/vacA was transfected into macrophages. The cytokine content of TNF-α or IL-1β in the culture medium was tested quantitatively with ELISA kit, respectively. The content of NO or ROS in the culture medium was tested with Griess reagent or DCFH-DA fluorescent probe. The apoptosis rate of macrophages was tested by flow cytometry. The effect of PDTC, an inhibitor of NF-κB, on the secretion and apoptosis of macrophages transfected with the recombinant plasmids, was also studied. The activity of NF-κB was examined in THP-1 cells by electrophoretic mobility gel shift assay（EMSA）. Resuits At 6 h after transfection, the level of TNF-α and IL-1β in macrophages transfected with the recombinant plasmids was significantly higher than that of the control group （ P 〈 0. 05 ）. At 6 h or 12 h after transfection, the level of NO and ROS in macrophages transfected with the recombinant plasmids was significantly higher than that of the control group （ P 〈 0. 05 ）. At 16 h after transfection, the apoptosis rate of macrophages transfected with the recombinant plasmids was significantly higher than that of the control group （ P 〈 0.05）. PDTC decreased the production of TNF-α, IL-1β, NO, ROS and apoptosis rate induced by VacA. VacA was found to trigger NF-κB activation. Conclusion The over-expression of VacA fusion protein can up-regulate secretion and apoptosis of macrophages. Activation of NF-κB is probably involved in the production of TNF-α, IL-1β, NO, ROS and apoptosis induced by VacA.