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目的:探讨MRP8刺激星形胶质细胞(astrocyte,AS)释放IL-1β的信号途径。方法:利用MRP8刺激培养AS,运用Lipo2000脂质体转染发卡样小干扰RNA(shRNA)使TLR4沉默、二硫代氨基甲酸吡咯烷(PDTC)预处理阻断NF-κB,运用免疫印迹(Western Blot)、电泳迁移率变动分析(EMSA)、酶联免疫吸附试验(ELISA)、间接荧光免疫双标法等实验方法检测TLR4、NF-κB以及IL-1β的表达变化及相互作用关系。结果:利用MRP8刺激培养AS,细胞内的TLR4、NF-κB以及IL-1β表达增加,并且NF-κB由胞浆向胞核内转移,IL-1β在细胞上清液中含量增加;抑制TLR4可以下调MRP8所致的NF-κB和IL-1β表达增加,NF-κB向胞核内转移;抑制NF-κB只能下调MRP8所致的IL-1β表达增加。结论:MRP8可能通过TLR4/NF-κB信号途径促进刺激星形胶质细胞分泌IL-1β。
AIM: To investigate the signaling pathways by which MRP8 stimulates IL-1β release from astrocytes (AS). Methods: AS was stimulated with MRP8 and silenced TLR4 by Lipo2000 liposome-transfected shRNA. NF-κB was blocked by pyrrolidine dithiocarbamate (PDTC) pretreatment. Western blotting was performed by Western blotting Blot, electrophoretic mobility shift assay (EMSA), enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence double labeling were used to detect the expression and interaction of TLR4, NF-κB and IL-1β. Results: The expression of TLR4, NF-κB and IL-1β in the cells were increased by MRP8 stimulation, and NF-κB was translocated from the cytoplasm to the nucleus and the content of IL-1β in the supernatant increased. MRP8 can downregulated the expression of NF-κB and IL-1β, NF-κB transfer to the nucleus; inhibition of NF-κB can only reduce MRP8-induced IL-1βexpression increased. Conclusion: MRP8 may promote the secretion of IL-1β by astrocytes through the TLR4 / NF-κB signaling pathway.