摘要
目的 探讨晚期糖基化终产物 (AGE)诱导单核细胞产生白细胞介素 1β(IL 1β)和肿瘤坏死因子 浕(TNF 浕)的细胞内信号传导机制。方法 采用密度梯度离心法分离健康成人外周血单核细胞 ,经AGE修饰的人血清白蛋白 (AGE HSA)刺激后 ,用ELISA法检测培养上清中IL 1β、TNF 浕水平 ,用化学发光法测定活性氧的生成 ;免疫细胞化学染色及凝胶迁移电泳 (EMSA)法观察核因子 κB(NF κB/p6 5 )的激活。分别用抗AGE受体 (RAGE)抗体、NADPH氧化酶特异性抑制剂apocynin和p38通路抑制剂SB 2 0 35 80预处理单核细胞 ,观察对AGE HSA诱导细胞因子和活性氧产生的影响。结果 AGE HSA与单核细胞在体外共同培养后 ,细胞内NF κB激活 ,培养上清中IL 1β、TNF 浕水平明显增高 ,活性氧生成增加 ;用抗RAGE抗血清或apocynin预处理细胞可阻断AGE HSA诱导的活性氧生成 (P <0 0 1) ,抑制NF κB的活化并使培养上清IL 1β、TNF 浕显著降低 (P <0 0 1)。用SB 2 0 35 80预处理细胞也可抑制AGE HSA诱导的NF κB激活及IL 1β、TNF 浕的产生 ,但对活性氧的产生无明显影响 (P >0 0 5 )。结论 AGE通过RAGE介导的途径刺激单核细胞生成IL 1β、TNF 浕和活性氧 ,NADPH氧化酶途径可能是RAGE细胞内信号途径的上游 。
Objective To investigate the cellular receptor pathway and the intracellular signaling of advanced glycation end products(AGE)-induced inflammatory reaction in monocytes. Methods Human peripheral monocytes were isolated from healthy volunteers. Cells were incubated with AGE modified by the addition of human serum albumin (AGE-HSA) either with pretreatment or no pretreatment of anti-AGE receptor (RAGE) IgG, NADPH oxidase inhibitor (apocynin)or a specific inhibitor of p38(SB 203580). The levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α) in the supernatants were assayed with enzyme-linked immunoadsorbent assay (ELISA). Reactive oxygen species (ROS) production was determined by MCLA chemiluminescence. Nuclear factor-κB translocation was assayed by immunochemical staining with anti-NF-κB/p65 and electrophoretic mobility shift assay(EMSA). Results AGE-HSA was found to induce activation of NF-κB, increase levels of IL-1β and TNF-α in the supernatants, and enhance production of ROS by monocytes. Pre-treatment of cells with anti-RAGE IgG or apocynin inhibited AGE-HSA to induce NF-κB translocation and IL-1β or TNF-α production. AGE stimulated ROS production could also be blocked by pre-treatment of cultured cells with anti-RAGE IgG or apocynin. Pre-treatment of cultured cells with SB 203580 inhibited both NF-κB activation and cytokines production, but showed no significant effect the cells to produce ROS. Conclusion AGE-HSA could induce IL-1β and TNF-α release as well as ROS production in human monocytes via a pathway mediated by RAGE. Activation of NADPH oxidase may be the upstream of the intracellular pathway. AGE-induced cytokines production was p38 pathway-dependent.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2004年第11期947-950,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学重点项目基金(编号 30 330 30 0 )
广东省团队项目(编号 1 0 71 7)资助课题
