高糖作用的肾小球系膜细胞SnoN/Ski蛋白表达及泛素化降解

目的: 观察高糖作用的大鼠肾小球系膜细胞内核转录共抑制因子SnoN/Ski及泛素连接酶Arkadia的表达变化,初步探讨SnoN/Ski泛素化降解在糖尿病肾病中的作用。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 、高糖甲组(培养基含20 mmol/L葡萄糖)、高糖乙组(培养基含30 mmol/L葡萄糖)、高糖+MG132组(30 mmol/L葡萄糖培养基中预先加入0.5 μmol/L特异性泛素蛋白酶体抑制剂MG132)和甘露醇组。用蛋白免疫印迹技术和免疫荧光染色-激光共聚焦显微镜检测各组细胞SnoN/Ski及Arkadia蛋白的表达。结果: (1)正常系膜细胞中SnoN/Ski蛋白大量表达,Arkadia蛋白表达较弱。(2)高糖作用后SnoN/Ski蛋白表达减弱,Arkadia蛋白表达增强(P<0.05)。(3)与高糖组比较,高糖加入MG132后SnoN/Ski蛋白表达增加,Arkadia蛋白表达减弱(P<0.01)。(4)甘露醇组与正常组比较SnoN/Ski及Arkadia蛋白表达均无明显差异(P>0.05)。结论: (1)高糖可通过泛素蛋白酶体途径降解SnoN/Ski蛋白致其低表达。(2)E3连接酶Arkadia参与了高糖诱导的SnoN/Ski的泛素化降解 。

Ubiquitin-dependent degradation of SnoN/Ski protein in glomerular mesangial cells induced by high glucose

AIM:To observe the protein expression of SnoN/Ski and ubiqutin ligase Arkadia in rat glomerular mesangial cells(GMCs) exposed to the high glucose.METHODS:Cultured rat glomerular mesangial HBZY-1 cells were divided into control group,20 mmol/L glucose group,30 mmol/L glucose group,30 mmol/L glucose + MG132 group (culture medium containing 30 mmol/L glucose and 0.5μmol/L specific proteasome inhibitor MG132),and mannitol group.The expression levels of SnoN,Ski and Arkadia were measured by Western blotting analysis,immunofluorescence and laser scanning confocal microscopy.RESULTS:In control GMCs,the expression of SnoN/Ski was rich and Arkadia was weak.After stimulated with high glucose,the expression of SnoN/Ski was decreased and Arkadia was gradually increased (P<0.05).Compared with high glucose group,the levels of SnoN/Ski and Arkadia were mostly reverted by adding the proteasome inhibitor MG132 at concentration of 0.5μmol/L(P < 0.01 ).The expression levels of SnoN/Ski and Arkadia were not significantly changed in mannitol group in comparison with control group(P > 0.05 ).CONCLUSION: High glucose decreases the expression of SnoN/Ski through ubiquitin-dependent degradation of SnoN/Ski.The degradation of SnoN/Ski mediated by Arkadia may play an important role in the pathogenesis of diabetic nephropathy.