晚期糖基化产物通过RAGE/TLR4/STAT1信号通路诱导巨噬细胞M1型极化  

Advanced glycation end products induces macrophage M1 polarization via RAGE/TLR4/STAT1 signaling pathway

作　　者：汤祥瑞 张勇[2] 祝领[2] 崔倩卫[2] 刘仲伟[2] 石爽[2] Tang Xiangrui;Zhang Yong;Zhu Ling(Dept of Cardiology,3201 Hospital,Hanzhong 723000;Dept of Cardiology,Shaanxi Provincial People′s Hospital,Xian 710068)

机构地区：[1]三二〇一医院心血管内科,汉中723000 [2]陕西省人民医院心血管内科,西安710068

出　　处：《安徽医科大学学报》2021年第5期751-756,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81700401);中国博士后科学基金特别资助项目(编号:2017T100760)。

摘　　要：目的探讨晚期糖基化产物(AGEs)诱导巨噬细胞发生M1型极化的分子机制。方法提取小鼠骨髓细胞并分离培养原代M0型巨噬细胞。分别用Toll样受体4(TLR4)及AGEs受体(RAGE)特异性抑制剂TAK-242及FPS-ZM1对获得的M0型巨噬细胞进行预处理。制备AGEs并以2.5、5及10μmol/L浓度处理巨噬细胞。流式细胞术检测细胞内活性氧簇(ROS)水平;免疫荧光染色观察M1型巨噬细胞表面标记物诱导性一氧化氮合酶(iNOS)表达;酶联免疫吸附试验法对巨噬细胞分泌的炎性因子浓度进行检测;Western blot检测相关浆蛋白及核蛋白的相对表达水平。结果AGEs刺激可使巨噬细胞内ROS水平、分泌炎性因子浓度、细胞质TLR4、磷酸化信号转导与转录激活因子1(p-STAT1)以及STAT1核转位水平显著升高(P均<0.001),巨噬细胞表面iNOS表达水平显著升高(P均<0.001),且均具有显著的AGEs浓度依赖性(P均<0.001)。TAK-242预处理对AGEs诱导的巨噬细胞iNOS表达,细胞内ROS水平及TLR4表达水平无显著影响,可显著降低AGEs诱导的巨噬细胞i NOS表达,分泌炎症因子浓度(P均<0.001)、细胞pSTAT1表达水平及STAT1核转位水平(P均<0.001)。FPSZM1预处理可显著抑制AGEs诱导的巨噬细胞内ROS水平(P<0.001)、分泌炎症因子浓度(P均<0.001)、细胞TLR4及p-STAT1表达水平(P均<0.001)以及STAT1核转位水平(P<0.001)。结论AGEs可通过RAGE/ROS/TLR4/STAT1信号通路诱导巨噬细胞向M1型极化。Objective To investigate the molecular mechanisms of advanced glycation end products(AGEs)-induced macrophage M1 polarization. Methods Primary M0 macrophages were isolated from mice bone marrow. Specific inhibitors of Toll-like receptor 4(TLR4) and receptors for AGEs(RAGE), namely TAK-242 and FPS-ZM1 were used to pre-treat the macrophages. Then the macrophages were exposed to AGEs at 2.5, 5 and 10 μmol/L respectively. Flow cytometry was used to detect intracellular reactive oxygen species(ROS) levels;immumofluorescence staining was used to observe the expression of M1 macrophage phenotype hall marker inducible nitric oxide synthase(iNOS);ELISA was used to determine the concentrations of inflammatory cytokines secreted from macrophages;Western blot was used to evaluate relative expression levels of cytosol and nuclear proteins.Results AGEs treatment induced elevation of intracellular ROS levels, interleukin 6(IL6), IL12 and tumor necrosis factor α(TNFα) concentrations, relative expression levels of cytosol TLR4, phosphorylated signal transducers and activators of transcription 1(p-STAT1) and nuclear STAT1 expressions in macrophages in a concentration dependent manner(all P<0.001). Pre-treatment of TAK242 significantly reduced iNOS expression, inflammatory cytokine concentrations, p-STAT1 expression and STAT1 nuclear translocation in AGEs-treated macrophages in a concentration dependent manner(all P<0.001) without affecting TLR4 expression and intracellular ROS levels. Pre-treatment of FPS-ZM1 significantly reduced iNOS expression, intracellular ROS levels(P<0.001), inflammatory cytokine concentrations(all P<0.001), TLR4 and p-STAT1 expressions(all P<0.001) and STAT1 nuclear translocation(P<0.001) in AGEs-treated macrophages. Conclusion AGEs can induce macrophage M1 polarization via RAGE/ROS/TLR4/STAT1 signaling pathway.

关 键 词：晚期糖基化产物 巨噬细胞 极化 TOLL样受体4 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学]