MAPKs抑制剂对大鼠肝细胞GSH代谢相关酶的影响

摘要

Objective:To investigate the effects of mitogen-activated protein kinases (MAPKs) inhibitors on glutathione (GSH) metabolism,and to explore the pathway related to GSH metabolism.Methods:BRL rat hepatocytes were treated by c-Jun NH2-terminal kinase (JNK),p38,and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors:SP600125,SB203580 and PD98659,respectively,for 24 h.MTT method was used to measure hepatocytes viability.The content of GSH was determined by high performance liquid chromatography.The protein expressions of JNK and phosphorylated JNK (p-JNK) was tested by Luminex method.Activities of GSH metabolic enzymes were detected by commercial kits.Results:Hepatocytes vitality was inhibited when the concentrations of SP600125,SB203580 and PD98659 were higher than 10 μmol/L,20 μmol/L,and 40 μmol/L,respectively;SP600125 decreased the content of GSH in hepatocytes,while SB203580 and PD98659 had no effect.SP600125 reduced p-JNK protein expression,and enhanced GSH-Px activity significantly.Conclusion:JNK MAPK pathway takes part in the GSH metabolism in hepatocytes.%目的:观察丝裂原活化的蛋白激酶(MAPKs)抑制剂对大鼠肝细胞谷胱甘肽(GSH)代谢的影响,确定哪条途径与GSH代谢相关.方法:体外培养BRL大鼠肝细胞,以c-Jun NH2-末端激酶(JNK)途径抑制剂SP600125、p38途径抑制剂SB203580、细胞外信号调节激酶1/2(ERK1/2)途径抑制剂PD98659处理24h,采用MTT法测定细胞活力,高效液相色谱法测定细胞内GSH含量,Luminex法测定JNK和磷酸化JNK(p-JNK)的蛋白表达,采用试剂盒测定GSH代谢酶活性.结果:SP600125浓度＞5μmol/L,SB203580浓度＞20 μmol/L,PD98659浓度＞40 μmol/L时,细胞活力受抑制;SP600125能显著减少大鼠肝细胞内还原型GSH的含量,SB203580和PD98659作用不明显;SP600125显著减少磷酸化JNK(p-JNK)蛋白表达,显著增强谷胱甘肽过氧化物酶(GSH-Px)的活力.结论:JNK MAPK途径参与了大鼠肝细胞GSH的代谢.