丙丁酚抑制氧化型低密度脂蛋白诱导系膜细胞转化生长因子-β_1表达

Probucol Inhibits Transforming Growth Factor-β_1 Expression Induced by Oxidized LDL in Rat Mesangial Cells

目的 观察丙丁酚对ox- LDL诱导系膜细胞AP -1活性和TGF -β1 表达的作用 ,探讨抗氧化剂治疗脂质肾损伤的分子机制。方法 大鼠系膜细胞随机分为正常细胞组、ox- LDL处理组和ox- LDL +丙丁酚处理组。运用凝胶迁移率实验检测AP-1活性变化 ,Western杂交检测c- Jun、JNK/SAPK磷酸化及TGF- β1 蛋白表达水平的改变。运用Northern杂交检测TGF- β1- mRNA表达。结果 丙丁酚 5 0 μmol/L预处理系膜细胞 2 0h ,显著降低ox LDL诱导系膜细胞AP 1活性。丙丁酚浓度为 5 0、1 0 0、2 0 0 μmol/L时 ,均显著抑制ox- LDL诱导JNK ,/SAPK磷酸化 ;且ox LDL +丙丁酚处理组的c Jun蛋白表达分别为ox- LDL处理组的 6 0 %、5 1 %和 4 6 %。Northern杂交显示 :ox LDL为 1 0 μg/mL时并不激活TGF- β1 mRNA表达 (P >0 .0 5 ) ;而当ox-LDL浓度增至 5 0、1 0 0 μg/mL时 ,TGF -β1 mRNA表达分别增加 1 .8和 1 .9倍。Western杂交显示ox -LDL呈剂量依赖方式增加TGF β1 蛋白质表达。加入丙丁酚后显著降低ox LDL诱导系膜细胞的TGF -β1- mRNA和蛋白质表达 (P <0 .0 5 )。结论 丙丁酚可能通过抑制JNK1 /SAPK磷酸化和AP 活性下调ox- LDL诱导系膜细胞TGF- β1 表达。

Purpose To investigate the effects of probucol on AP-1 binding activity and expression of transforming growth factor β 1(TGF-β 1) by oxidized LDL in mesagngial cells and clarify the possible molecular mechanisms of antioxidant in lipid-induced glomerular injury. Methods Rat mesangial cells were randomized to divided into three groups:normal cells,ox-LDL-treated cells and probucol+ox-LDL-treated cells.AP-1 binding in mesangial cells was measured by gel shift assay.The protein expression of c-Jun and TGF-β 1,and the phosphorylation of JNK/SAPK were detected by Western blot analysis.The expression of TGF-β 1 mRNA was detected by Northern blot analysis. Results A 20-hour treatment with probucol (50 μmol/L) was applied prior to 24-hour incubation of ox-LDL in mesangial cells.It was found that AP-1 binding had no difference between probucol-treated normal cells and normal ones,but probucol at 50 μmol/L appreciably reduced the ox-LDL-induced AP-1 binding.Probucol at 50,100,200 μmol/L significantly inhibited the phosphorylation of JNK 1/SAPK after being exposed to ox-LDL(100 μg/mL) for 30 min in mesangial cells.Probucol at 50,100,200 μmol/L reduced c-Jun expression induced by ox-LDL.Ox-LDL at 10 μg/mL did not stimulate TGF-β 1 mRNA expression,but when ox-LDL rose to 50,100 μg/mL,TGF-β 1 mRNA expression increased 1.8 and 1.9 times,respectively.Simultaneously,ox-LDL stimulated the expression of TGF-β 1 protein markedly.After being exposed to probucol,the ox-LDL-induced expression of TGF-β 1 mRNA and protein decreased significantly (P<0.05). Conclusions Probucol downregulates the expression of TGF-β 1 mRNA and protein induced by ox-LDL in mesangial cells,possibly through the inhibition of the phosphorylation of JNK 1/SAPK and AP-1 binding activity.