氧化低密度脂蛋白诱导Zucker大鼠肾小球系膜细胞中NF-κB活性的变化

Enhance effect of Ox-LDL on NF-κB activity in glomerular mesangial cells from the Zucker rats

目的 :研究氧化低密度脂蛋白 (Ox LDL)对体外培养的Zucker大鼠肾小球系膜细胞 (GMC)核因子κB (NF κB)活性的影响以及NF κB的活性变化与大鼠鼠龄及基因型的相关性。方法 :(1)将 3月龄和 10月龄Zucker肥胖大鼠及Zucker瘦型大鼠的GMC(O3m、O10m,、L3m和L10m)进行传代培养。 (2 )采用电泳迁移率变动分析 (EMSA)和超迁移率实验 (GSA) ,检测不同浓度及不同时间相的Ox LDL对GMC中NF κB活性的影响及其亚单位p5 0和p6 5的变化。利用Westernblot检测Ox LDL刺激后NF κB的核转位。结果 :Ox LDL刺激后 ,O3m、O10m及L3m3组GMC中NF κB的活性均明显强于对照组 (P <0 .0 1) ;L10m组与对照组相比较无显著差异 (P >0 .0 5 )。随着Ox LDL浓度的增加和刺激时间的延长 ,GMC中NF κB活性也相应增强 ,5 0mg/L的Ox LDL刺激 4h时 ,NF κB的活性强度达最高峰。Ox LDL主要激活NF κB的p6 5及p5 0亚单位 ;各组NF κB的活性相比较 :O3m 组高于O10m组 ,O3m组高于L3m组及O10m组高于L10m组 (P均 <0 .0 1) ;L3m组NF κB的活性高于L10m组 (P <0 .0 5 ) .结论 :Ox LDL可诱导Zucker大鼠GMC中NF κB活化 ,且呈时间和浓度依赖性 ;活性强度与大鼠的基因型及鼠龄密切相关。Ox LDL诱导的NF κB活化在Zucker肥胖大鼠的早期肾损害中起着更为重?

AIM: To study the effect of oxidized low-density lipoprotein (Ox-LDL) on nuclear NF-κB activity in cultured glomerular mesangial cells (GMCs) from the Zucker rats and the correlation between the change of NF-κB activity and ras’s age and genotype. METHODS: Four groups of GMCs (O 3m, O 10m, L 3m and L 10m) from the 3- and 10- month (fa/fa) and 3- and 10- month lean (Fa/Fa) Zucker rats were stimulated with Ox-LDL. Electrophoretic mobility shift assay (EMSA) and Western blot were used to detect the activity of NF-κB. Gel supershift assay(GSA) was used to detect the subunit of NF-κB dimer. RESULTS: (1) NF-κB activity after induction with Ox-LDL in the 3 groups (O 3m, O 10m and L 3m) of GMCs were significantly higher than that in the control group (P<0.01). (2) With the increased of concentration and stimulation time of Ox-LDL, the NF-κB activity was increased correspondingly and reached the highest when GMCs were stimulated with 50 mg/L Ox-LDL for 4 hours. (3) Supershift assay demonstrated that Ox-LDL mainly activated the two subunits p65 and p50 in GMCs. (4) As the activity of NF-κB, O 3m group vs O 10m group, O 3m group vs L 3m group, and O 10m group vs L 3m group had all markedly differences (P<0.01), while the differences between L 3m group vs L 10m group was inferior to 3 groups above (P< 0.05). COCLUSION: Ox-LDL could significantly activate NF-κB in GMCs from Zucker rats, in the time- and dose- dependence manner. There was a close relationship between the NF-κB activity in GMCs induced with Ox-LDL, the age and genotype of Zucker rats. NF-κB activity in GMCs of the Zucker rats induced with Ox-LDL plays an important role in the early renal lesion of diabetes mellitus.