胰岛素诱导Zucker肥胖大鼠肾小球系膜细胞核因子-κB活性的变化

目的 研究胰岛素(INS)对体外培养的Zucker大鼠肾小球系膜细胞(GMC)核因子κB(NF-κB)活化的影响,以及INS诱导的NF-κB活性的变化与Zucker大鼠年龄(即病程)以及基因型的相关性。方法 (1)分别取Zucker肥胖大鼠(3月龄和10月龄)和Zucker瘦型大鼠(3月龄和10月龄)4组大鼠的GMCs(O_(3m),O_(10m),L_(3m),L_(10m))进行传代培养。(2)凝胶迁移率实验(EMSA)和超迁移率实验(GSA)检测不同浓度及不同时相INS对Zucker大鼠肾小球系膜细胞NF-κB活性的影响,以及NF-κB二聚体中p50和p65成分的变化。(3)应用Western印迹检测胞浆和胞核内NF-κB p65水平。结果 (1)INS诱导后,4组GMC内NF-κB活性均较对照组明显增强(F=219.65,P<0.01);(2)随着INS刺激浓度增加和时间的延长,GMC内NF-κB活性相应增强,0.5 mg/L的INS刺激24h时,NF-κB活性强度达最高峰;INS刺激浓度升为5 mg/L,则15h达最高峰;(3)INS主要激活NF-κB二聚体成份中的p65;(4)O_(3m)组NF-κB的活性显著高于O_(10m)组(P<0.01),L_(3m)组NF-κB的活性显著高于L_(10m)组(P<0.01),O_(3m)组显著高于L_(3m)组(P<0.01),O_(10m)组显著高于L_(10m)组(P<0.01)。(5)INS可呈剂量依赖性地降低胞浆内的p65的蛋白水平和上调胞核内p65水平。结论 INS可诱导Zucker大鼠GMC内NF-κB活化,其活化方

Enhance effect of insulin on nuclear facteor-kappa B activation in glomerular mesangial cells of the Zucker rats

Objective To investigate the effect of insulin (INS) on nuclear factor-kappa B (NF-KB) activation in glomerular mesangial cells(GMC) of the Zucker rats,and the correlation between the activity of NF-kB induced by insulin to the ages and genotype of Zucker rats. Methods (1) Four groups of cultured GMCs(O3m,O10m,L3m and L10m) from the Zucker obese rats(3 months old and 10 months old) and Zucker lean rats(3 months old and 10 months old) were stimulated by insulin. (2) Electrophoretic mobility shift assay (EMSA) was used to detect the activity of NF-KB. Gel supershift assay was used to detect the subunit of NF-KB dimer. (3) The protein of NF-KB p65 in cytoplasm and cytoblast was analysed by Western Blot. Results (1) NF-KB activity in 4 groups GMCs was significantly higher than that in control group after induced of INS ( F=219. 65 P < 0. 01);(2) With the increase of stimulation concentration and time of INS,the NF-kB activity was risen accordingly. The level of NF-kB activity was highest when GMCs were stimulated by INS 0. 5 mg/L,24 h or 5 mg/L,15 h; (3) Supershift assay demonstrated that p65 was the predominant subunit involved. (4)The NF-KB activity in O3m was higher than in O10m( P < 0. 01),L3m was higher than L10m( P < 0. 01),O3mwas higher than L3m( P < 0. 01),O10m was also higher than L10m( P < 0. 01); (5) INS induced nuclear translocation of NF-kB p65 subunit,presenting time-dependent and dose-dependent manner. Conclusions INS significantly activates NF-kB in GMCs of Zucker rats in time- and dose-dependent manner. There is an intimated correlation between the activity of NF-kB induced by INS to the ages and genotype of Zucker rats. It plays an important role in the early renal lesion of diabetes mellitus that enhance effect of INS on NF-kB activation in GMCs of the Zucker rats.