氧化型低密度脂蛋白刺激小鼠足细胞后的氧化应激效应和机制研究

Effects of oxidized low-density lipoprotein on glomerular podocytes and its mechanisms

目的观察氧化型低密度脂蛋白(ox-LDL)刺激小鼠足细胞后的氧化应激效应和信号通路的激活,探讨足细胞抗氧化能力变化的机制。方法分别用50μg/ml ox-LDL、LDL刺激分化成熟的小鼠肾小球足细胞(ox-LDL刺激组、LDL刺激组),并设单纯以无血清培养基培养的足细胞为对照组,观察12、24 h足细胞的损伤情况及超氧化物歧化酶(SOD)活性的变化。采用TUNEL法判断足细胞的凋亡情况,免疫荧光法及Western blot法观察足细胞裂孔膜蛋白nephrin表达情况,间接免疫荧光法观察细胞骨架蛋白paxillin、F-actin的表达情况,Western blot法进一步检测两条信号通路丝裂原活化蛋白激酶(MAPK)、糖原合成酶激酶3β(GSK-3β)的变化,分光光度法测定足细胞细胞质SOD的活性。结果经LDL及ox-LDL刺激12、24 h的足细胞均未见凋亡,12 h 3组细胞nephrin的表达无明显差异,24 h ox-LDL刺激组nephrin表达明显下调、足突上的paxillin表达消失,F-actin重新分布。12 h ox-LDL刺激组及LDL刺激组的MAPK和GSK-3β活性无明显差异,24 h ox-LDL刺激组磷酸化的MAPK和GSK-3β比率显著增加。12 h ox-LDL刺激组足细胞SOD活性较对照组增加19.94%,ox-LDL刺激持续至24 h,SOD活性较对照组仅增加7.3%。结论50μg/ml ox-LDL刺激体外培养的分化成熟小鼠肾小球足细胞24 h,可导致nephrin显著下调,细胞骨架蛋白重构,磷酸化的MAPK、GSK-3β比率显著增加,提示MAPK、GSK-3β两条通路的激活可能导致nephrin表达下调和细胞骨架蛋白重构。氧化刺激早期可造成足细胞的SOD活性反应性升高并使足细胞免于氧化损伤。

Objective To investigate the effects of oxidized low density lipoprotein(ox-LDL)on differentiated mouse podocytes and its mechanism.Methods The differentiated mouse podocytes were stimulated with 50μg/ml ox-LDL and low density lipoprotein(LDL)respectively,and the serum-free culture medium was used in the control group.The damage of podocytes and the activity of superoxide dismutase(SOD)were observed at 12 and 24 h of treatment.The apoptosis of podocytes was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)kit.Indirect immunofluorescence was used to observe the expression of slit diaphragm protein nephrin and cytoskeleton proteins paxillin and F-actin;protein levels of nephrin,mitogen-activated protein kinase(MAPK)and glycogen synthase-3β(GSK-3β)involved in the injury were semi-quantified with Western blot,and spectrophotometry was used to determine the SOD activity of podocytes.Results There was no apoptosis in podocytes at 12 h and 24 h of treatment in all 3 groups.There was no difference in the expression of nephrin in LDL and ox-LDL stimulation groups at 12 h.The expression of nephrin decreased significantly in ox-LDL stimulation group at 24 h.The expression of paxillin was negative and F-actin was redistributed in ox-LDL stimulation group at 24 h.The activity of MAPK and GSK-3β did not change significantly in the two groups at 12 h,and the ratio of phosphorylated MAPK and GSK-3β increased significantly in the ox-LDL stimulation group at 24 h.The SOD activity of podocytes stimulated by ox-LDL was 19.94% higher at 12 h of treatment and 7.3% higher at 24 h of treatment than that in the control group,respectively.Conclusion The treatment of ox-LDL leads to the down-regulation of nephrin expression and cytoskeleton protein reconstruction in mouse podocytes,which may be associated with the activation of MAPK and GSK-3β pathways.In the early stage of oxidative stimulation,the increase of SOD activity may protect podocytes fromoxidative damage.