MG132对高糖刺激的肾系膜细胞中Smad7表达的作用

Effect of MG132 on expression of Smad7 in cultured glomerular mesangial cells induced by high concentration of glucose

目的观察不同浓度高糖刺激后大鼠肾系膜细胞胞内Smad7的表达,并以蛋白酶体特异性抑制剂MG132作为阻断剂,探讨泛素化降解在Smad信号中的作用。方法将体外培养的大鼠肾小球系膜细胞分别设正常对照组(葡萄糖浓度5.6mmol/L)、20 mmol/L高糖组、30 mmol/L高糖组、30 mmol/L高糖+MG132组、甘露醇组、正常对照组+MG132、30 mmol/L高糖+溶剂组等。分别用Real ti me Quantitative PCR法和细胞免疫荧光染色法及激光共聚焦显微镜检测各组细胞Smad7的mR-NA和蛋白的表达。结果(1)与正常对照组比较,高糖作用后,各组Smad7 mRNA的表达差异无统计学意义;(2)而正常对照组细胞Smad7蛋白表达较强;高糖组较正常对照组表达减弱(P<0.05),呈浓度依赖性,30 mmol/L高糖组加入MG132后,Smad7蛋白表达增强。结论(1)高糖可诱导肾系膜细胞Smad7蛋白降解增加;(2)MG132可阻止高糖所导致的上述变化。提示泛素-蛋白酶体途径参与了糖尿病肾病Smad通路的信号调节。

Objective To observe the expression of Smad7 in rat glomerular mesangial cells（GMC） stimulated by the high glucose of different concentration,and to investigatiethe the effect of the ubiquition in this smad signal by adding MG132 as a proteasome differential inhibitor.Methods Cultured rat GMC were divided into normal group（the concentration of glucose：5.6 mmol/L）,high glucose group（20 mmol/L,30 mmol/L respectively）,therapy group（30 mmol/L high glucose with MG132）,mannital group,normal glucose＋MG132 group,30 mmol/L glucose＋solvent group.The expression of Smad7 mRNA of each group was measured by quantitative PT-PCR and protein was measured by indirect immunfluorescence and laser scanning confocal microscope respectively.Results（1）Compared with normal control group,the expression of Smad7 mRNA had no significant change.（2）The expression of Smad7 protein was decreased significantly in a dose dependent manner in high glucose（P〈0.05）;in therapy group,the expression of Smad7 protein was enhanced.Conclusion（1）High glucose can increase the ubiquitinated degradation of Smad7 protein in glomerular mesangial cells.（2）Compared with 30 mmol/L glucose group,the degradation of Smad7 protein could be reverted mostly by MG132（P〈0.05）.Ubiquition-proteasome pathway（UPP） is related with the regulation of Smad signal transduction pathways in diabetic nephropathy（DN）.