丙丁酚对系膜细胞转录激活蛋白-1活性的影响

PROBUCOL INHIBITS ACTIVATOR PROTEIN 1 BINDING INDUCED BY OXIDIZED LDL IN RAT MESANGIAL CELLS

目的 :研究氧化型低密度脂蛋白 (ox LDL)诱导肾小球系膜细胞转录激活蛋白 1 (AP 1 )活性的改变及抗氧化剂丙丁酚对其影响 ,进一步探讨丙丁酚抗氧化作用的分子机制。 方法 :利用凝胶迁移率实验 (Gelshiftas say)和超迁移率实验 (Gelsupershiftassay)检测不同浓度及不同时相ox LDL对大鼠肾小球系膜细胞AP 1活性的影响、AP 1二聚体中c Jun和c Fos成分的变化及抗氧化剂丙丁酚对其影响。 结果 :ox LDL(1 0 0mg/L)作用系膜细胞后 6h ,核蛋白提取物中AP 1活性开始升高 ,36h达到最大值 ,48h与 36h相比较无明显变化 ;ox LDL作用 2 4h时 ,随着ox LDL浓度的增加 ,AP 1活性逐渐增加 ;超迁移率实验显示ox LDL(1 0 0mg/L)主要激活AP 1二聚体成分中c Jun ,而Fos蛋白未激活 ;丙丁酚 (5 0nmol/L)对一般培养状态下系膜细胞AP 1活性无明显抑制作用 ,但能明显抑制ox LDL(1 0 0mg/L)诱导的系膜细胞AP 1活性。 结论 :丙丁酚降低ox LDL诱导的系膜细胞AP

OBJECTIVE To investigate the binding activity of transcription factor activator protein 1(AP 1) induced by oxidized low density lipoprotein(ox LDL) in rat mesangial cells and further to clarify the molecular mechanism of antioxidant probucol in the treatment of atherosclerosis. METHODOLOGY Human serum LDL was separated by sequencial ultracentrifugation and ox LDL was prepared by use of 5 μmol/L CuSO 4 oxidation. The rat mesangial cells were exposed to ox LDL, nuclear protein was extracted and the AP 1 binding was detected by gel shift assay. For supershifit assay, nuclear extracts were incubated with goat polyclonal antibodies against c Jun and pan Fos for 3 hours before adding the labeled AP 1 prob.Additionally, the effects of probucol on AP 1 binding activity of the control and the ox LDL treated mesangial cells were also examined. RESULTS After ox LDL(100 mg/L) was added in the medium of cultured mesangial cells, the binding activity of AP 1 began to rise at the 6th hour and reached the maximum at the 36th hour as compared with the control group. Moreover, no difference was found between the AP 1 binding activity at the 36th hour and that at the 48th hour.The AP 1 activity was stimulated when the rat mesangial cells were exposed to 25 mg/L ox LDL. With the increase of ox LDL concentrations, AP 1 binding activity of mesangial cells was found elevated. This ox LDL increased AP 1 binding involved c Jun, but not Fos as shown by gel supershift assay. Antioxidant probucol(50 nmol/L) inhibited the by ox LDL(100 mg/L) induced AP 1 activity in mesangial cells, but not in the control. CONCLUSION This study demonstrates that ox LDL stimulates AP 1 binding activity in time and dose dependent manners and probucol can inhibit transcription factor AP 1 activity induced by ox LDL in rat mesangial cells, which may be one of its antioxidation mechanisms in the treatment of atherosclerosis.