晚期糖基化产物受体信号传导在羧甲赖氨酸诱导足细胞表达单核细胞趋化因子中的作用

Effect of advanced glycation end product receptor signal transduction on MCP-1 expression of podocyte induced by carboxymethyllysine

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.