脂多糖诱导小鼠腹腔巨噬细胞缺氧诱导因子-1α表达的信号通路研究

Study on Signaling Pathways in HIF-1α Expression Induced by LPS in Murine Peritoneal Macrophages

目的研究体外常氧环境下脂多糖(LPS)诱导小鼠腹腔巨噬细胞缺氧诱导因子-1α(HIF-1α)表达的相关信号通路。方法以LPS单独或联合NF-κB抑制剂柳氮磺胺吡啶(SSZ)、一氧化氮合酶(NOS)抑制剂L-单甲基精氨酸(L-NMMA)作用于BALB/c小鼠腹腔巨噬细胞。用逆转录聚合酶链反应(RT-PCR)法检测HIF-1αmRNA的变化,用免疫细胞化学法检测HIF-1α蛋白的变化,用Griess反应检测培养上清中亚硝酸盐浓度的变化。结果LPS作用于小鼠巨噬细胞10h后,HIF-1αmRNA水平无明显变化(P>0.05),HIF-1α蛋白水平则明显增加(P<0.05)。SSZ和L-NMMA组HIF-1α蛋白的表达则明显低于LPS组(P均<0.05)。与对照组相比,LPS可明显增加巨噬细胞NO的生成(P<0.05),SSZ则可明显抑制LPS诱导的NO的产生(P<0.05)。结论LPS可通过NF-κB及iNOS等转录后信号途径上调HIF-1α的表达。

Objective To investigate the signaling pathways in expression of HIF-1α in murine peritoneal macrophages induced by LPS.Methods The peritoneal macrophages from BALB/c mice were collected and treated with LPS combined with or without NF-κB inhibitor SSZ or NOS inhibitor L-NMMA.The level of HIF-1α mRNA was measured by RT-PCR.The expression of HIF-1α protein was determined by immunocytochemical method,and the level of nitrite in cultural supernatant was measured by Griess reaction.Results After the peritoneal macrophages being treated with LPS for 10h,the level of HIF-1α mRNA did not exhibit significant change,compared with that in the control group（P0.05）;however the level of HIF-1α protein expression in the LPS group was significantly higher than that in the control group（P0.05）.The expression levels of HIF-1α protein in the groups of SSZ and L-NMMA were significantly lower than that in the LPS group（P0.05）.Meanwhile,compared with that in the control group,LPS could significantly increase the production of nitrite（P0.05）.SSZ could significantly lower that effect induced by LPS（P0.05）.Conclusion LPS can up-regulate the expression of HIF-1α by post-transcription mechanisms via NF-κB and NOS pathways.