摘要
目的了解足细胞上晚期糖基化产物受体(RAGE)激活后的细胞内信号的传导途径。方法激光共聚焦显微镜观察细胞内反应性氧自由基(ROS)的产生。Western印迹方法检测足细胞内丝裂原激活的蛋白激酶(MAPK)家族磷酸化,RT-PCR方法检测单核细胞趋化因子-1(MCP-1)的mRNA表达。结果足细胞细胞核内存在基础性的ROS,AGE和羧甲赖氨酸(carboxymethyllysine, CML)分别诱导细胞浆内ROS增加2.2和2.6倍。N-乙酰-L-半胱氨酸(NAC)抑制基础性和诱导性ROS形成,RAGE中和抗体则完全抑制诱导性的ROS产生。NAC也可直接抑制CML以及外源性H_2O_2诱导的MCP-1的表达。使用CML刺激足细胞10 min后,磷酸化细胞外信号调节激酶(ERK)增加3.8倍。而CML刺激足细胞120 min仍没有发现磷酸化的p38MAPK和应激活化蛋白激酶(SAPK)/氨基末端激酶(JNK)表达或上词。使用NAC,7氨4三氟甲基香豆素(AFC)可以完全防止ERK磷酸化并抑制MCP-1的mRNA表达。PD98059阻断了ERK磷酸化却并不能完全抑制MCP-1的mRNA的表达。结论足细胞上RAGE激活后,通过ROS-p21Ras-ERK信号途径诱导足细胞表达MCP-1。
Objective To investigate the signalling events follow the activation of RAGE in podocytes. Methods AGE and CML generated dichloroflurescin-sensitive intracellular ROS were measured by confoeal microscopy. The activation of MAP kinases family were studied using Western blotting. MCP-1 mRNA expression was detected by semi-quantitative RT-PCR. Results Basal ROS was located in nucleus of starvation podoeytes. AGE and CML rapidly generated intracellular ROS in podoeytes. NAC pre-treated podoeytes suppressed basal and inducible ROS generation and antibody for RAGE suppressed the inducible ROS. Blockage of ROS induced by NAC suppressed the expression of CML and H2O2-induced MCP-1. Phosphorylated extracelltdar signal-regulated kinase （ERK） was found in CML incubated podoeytes at 10 rain and was prevented by NAC or AFC. PD98058 Pre-treated podoeytes partially inhibited the expression of CML-induced MCP-1 mRNA. No evidence were showed that p38 MAPK, SAPK/JNK, PI3K and PKC were involved in the signal trmasduction. Conclusion Activation of RAGE induces MCP-1 expression in podoeytes via ROSERK signalling pathway.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2005年第11期689-694,共6页
Chinese Journal of Nephrology
关键词
单核细胞化学吸引蛋白1
糖基化终产物
高级
活性氧
有丝分裂素激活蛋白激酶类
足细胞
Monocyte chemoattractant protein 1
Glycosylation end products, advanced
Reactive oxygen species
Mitogen-activated protein kinases
Podocyte
