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目的探讨醛固酮对肾小球系膜细胞自噬激活的影响。方法通过3种经典的自噬活性检测方法检测醛固酮对体外培养的系膜细胞自噬的影响:(1)蛋白质印迹法检测不同浓度的醛固酮干预肾小球系膜细胞系(HMCL)后自噬相关的蛋白标记物LC3、SQSTM1/P62的表达水平变化;(2)向系膜细胞系转入GFP-LC3的真核表达质粒,用激光共聚焦显微镜观察醛固酮干预下系膜细胞荧光自噬点数量的变化并进行统计学分析;(3)通过电子显微镜观察醛固酮干预下系膜细胞自噬泡数量的变化并进行统计学分析。通过光镜下形态学观察及蛋白质印迹法检测凋亡相关蛋白聚腺苷二磷酸-核糖聚合酶(PARP)剪切体的表达情况,分析醛固酮干预下系膜细胞在过氧化氢刺激后凋亡水平的变化。结果 3种方法均证实高生理剂量醛固酮可以抑制系膜细胞自噬激活:(1)10-7 mol/L醛固酮干预12h后自噬标记蛋白LC3Ⅰ向LC3Ⅱ转化减少约40%;(2)10-7 mol/L醛固酮干预12h能够降低饥饿及雷帕霉素诱导的系膜细胞自噬点数量,降低的比率分别为60%和47%;(3)醛固酮干预后可减少饥饿和雷帕霉素诱导的系膜细胞自噬泡的增加。10-7 mol/L醛固酮干预系膜细胞12h后接受过氧化氢2.5×10-6 mol/L刺激较对照组细胞凋亡的比率明显增加(P<0.05)。结论高生理浓度的醛固酮能够抑制系膜细胞的基础自噬激活并促进系膜细胞在氧化应激条件下的凋亡。
Objective To investigate the effect of aldosterone on the activation of glomerular mesangial cells. Methods The effects of aldosterone on the autophagy of mesangial cells in vitro were tested by three classical methods of autophagy activity assay: (1) Western blotting was used to detect the effect of aldosterone on autophagy after intervention of mesangial cell line (HMCL) (2) The eukaryotic expression plasmid of GFP-LC3 was transfected into mesangial cell line, and the autophagy fluorescence of mesangial cells was detected by laser confocal microscopy (3) The change of autophagic vacuoles in mesangial cells under aldosterone intervention was observed by electron microscope and statistical analysis was made. Morphological observation and western blotting were used to detect the expression of apolipoprotein-ribose polymerase (PARP), the apoptosis of mesangial cells was observed after exposure to hydrogen peroxide. Horizontal changes. Results All three methods confirmed that high dose of aldosterone could inhibit the autophagy activation of mesangial cells: (1) The autophagy activation of mesangial cells was inhibited by 10-7 mol / L aldosterone for 12 h, and the conversion of LC3 Ⅱ to LC3 Ⅱ was reduced by about 40% 7mol / L aldosterone intervention 12h can reduce hunger and rapamycin-induced mesangial cell autophagy points the number of reduced rates were 60% and 47%; (3) aldosterone intervention can reduce hunger and rapamycin Induction of mesangial cell autophagy increased. The ratio of 10-7 mol / L aldosterone to mesangial cells exposed to 2.5 × 10-6 mol / L hydrogen peroxide for 12h increased significantly compared with control group (P <0.05). Conclusion The high concentration of aldosterone can inhibit the basic autophagy activation of mesangial cells and promote the apoptosis of mesangial cells under oxidative stress.