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(Gel shift as-say)和超迁移率实验(Gel supershift assay)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-Fcs成分的变化及抗氧化剂丙丁酚对其影响。结果：ox-LDL(100mg／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24h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(100mg／L)主要激活AP-1二聚体成分中c-Jun，而Fcs蛋白未激活；丙丁酚(50nmol／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(100mg／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-1活性可能是其作为抗氧化剂治疗动脉粥样硬化的分子机制之一。%OBJECTIVE To investigate the binding activity of transcription factor activator protein-1 (AP-1) induced by oxidized lowdensity lipoprotein(ox-LDL) in rat mesangial cells and further to clarify the molecular mechanism of antioxidant prolcol in thetreatment of atherosclerosis. METHODOLOGY Human serum LDL was separated by sequencial ultracentrifugation and ox-LDL was prepared by use of 5 μmol/L CuSO4 oxidation. The rat mesangial cells were exposed to ox-LDL, nuclear protein was ex-tracted and the AP-1 binding was detected by gel shift assay. For supershifit assay, nuclear extracts were incubated with goat poly-clonal antibodies against c-Jun and pan-Fos for 3 hours before adding the labeled AP-1 prob. Additionally, the effects of probucolon 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 andreached the maximum at the 36th hour as compared with the control group. Moreover, no difference was found between the AP-1binding activity at the 36th hour and that at the 48th hour. The AP- 1 activity was stimulated when the rat mesangial cells were ex-posed to 25 mg/L ox-LDL. With the increase of ox-LDL concentrations, AP-1 binding activity of mesangial cells was found elevat-ed. This ox-LDL-increased AP-1 binding involved c-Jun, but not Fos as shown by gel supershift assay. Antioxidant probucol(50nmol/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 in-hibit transcription factor AP-1 activity induced by ox-LDL in rat mesangial cells, which may be one of its antioxidation mechanisnsin the treatment of atherosclerosis.
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