细胞外信号调节激酶通路在儿童原发性肾病综合征激素耐药中的作用机制探讨

Extracellular signal-regulated kinase pathway is involved in steroid resistance in childhood nephrotic syndrome

目的探讨细胞外信号调节激酶（ERK）通路在儿童肾病综合征糖皮质激素耐药中的作用机制。方法①收集儿童肾病综合征激素耐药和激素敏感患儿外周血单个核细胞（PBMCs），Western-blot印迹法检测磷酸化ERK水平，激光共聚焦显微镜观察糖皮质激素受体GRa核转位情况，计算核浆比；②体外观察ERK抑制剂U0126对激素耐药患儿PBMCs GRa核转位的影响；③超抗原金黄色葡萄球菌肠毒素B型（SEB）体外诱导糖皮质激素抵抗模型中ERK活化时程；④观察ERK抑制剂U0126逆转超抗原SEB诱导的糖皮质激素抵抗作用；⑤观察ERK抑制剂U0126对激素抵抗细胞模型GRa核转位的影响。结果①激素耐药患儿PBMCs存在显著的ERK磷酸化以及糖皮质激素受体GRa核转位障碍；②ERK抑制剂U0126可以逆转激素耐药患儿PBMCs糖皮质激素受体GRa核转位障碍；③超抗原SEB体外诱导可以导致糖皮质激素抵抗发生，ERK抑制剂U0126后可解除SEB诱导的激素抵抗；④SEB作用1min后即出现p-ERK1／2，在24h仍维持在高水平表达。结论激素耐药患儿PBMCs存在显著的ERK活化，体外超抗原SEB刺激PBMCs后出现糖皮质激素抵抗，其作用机制显著持续的ERK活化，ERK抑制剂可逆转激素耐药患儿PBMCs及体外超抗原SEB诱导的糖皮质激素抵抗发生。

Objective To investigate the role of ERK pathway activation in the glucocorticoid resistant nephrotic syndrome in children. Methods ① Blood lymphocytes were collected with glucocorticoid resistant nephrotic syndrome and with glucocorticoid sensitive nephrotic syndrome, the levels of phospharilated ERK were measured by western-blot and nuclear translocation of GRα was observed and the ratios of nuclear/cytoplasmic GRα were quantitated by laser confocal flurescence microscope;② The effects of ERK inhibitor U0126 on nuclear translocation of GRα in blood lymphoeytes from children with glucocortieoid resistant nephrotic syndrome were observed; ③The time course of ERK activation was characterized in SEB superantigen-indueed glucocorticoid resistant lymphocytes; ④The attenuation of glucocorticoid resistance by ERK inhibitor U0126 and its effect on nuclear translocation of GRα in SEB superantigen-induced glucocorticoid resistant lymphocytes were observed. Resuits ①Significant stronger ERK activation was observed by western-blot in blood lymphocytes from children with glueocortieoid resistant nephrotic syndrome, and nuclear translocation of GRα was significantly inhibited in blood lymphocytes from children with glucocorticoid resistant nephrotic syndrome under laser confocal flurescence microscope; ②ERK inhibitor U0126 could significantly attenuate the inhibition of GRα nuclear transloeation in blood lymphocytes from children with glucocorticoid resistant nephrotic syndrome; ③Staphylococcal enterotoxin B （SEB） superantigen could induce glucocorticoid resistance in normal blood lymphocytes, demonstrated by the inhibition of GRα nuclear translocation. ERK inhibitor U0126 could significantly increase the GRα nuclear translocation and the ratios of nuclear/cytoplasmic GRα distribution; ④ERK activation was induced in normal blood lymphocytes 1 minute after exposure of SEB and remained prominent at 24 hours. Conclusion Sustained activation of ERK is involved in the generation of glucocorticoid resistance in childhood nephrotic syndrome. ERK inhibitor can restore glucocorticoid sensitivity in blood lymphocytes from children with glucocorticoid resistant nephrotic syndrome and SEB superantigen-induced in vitro glucocorticoid resistant lymphocytes.