醛固酮抑制肾小球系膜细胞的自噬激活加速其在氧化应激状态下的凋亡
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中国人民解放军济南军区第医院肾内科 青岛,中国人民解放军济南军区第医院肾内科 青岛,第二军医大学第二附属医院解放军肾脏病研究所 上海,第二军医大学第二附属医院解放军肾脏病研究所 上海,第二军医大学第三附属医院信号转导研究室 上海;、第二军医大学第三附属医院信号转导研究室 上海,第二军医大学第二附属医院解放军肾脏病研究所 上海,第二军医大学第二附属医院解放军肾脏病研究所 上海

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国家自然科学基金(81270765),总后勤部科技青年培育项目(I3QNP035).


Aldosterone inhibits autophagy activation of mesangial cells and accelerates cell apoptosis under oxidative stress condition
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    摘要:

    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12 h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12 h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12 h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。

    Abstract:

    Objective To investigate the effect of aldosterone on the activation of autophagy in mesangial cells. Methods Three classic techniques were used to detect the autophagy activity in mesangial cells with or without aldosterone treatment in this study. (1) Western blotting analysis was used to examine the expression of autophagy protein LC3, SQSTM1/P62 after human mesangial cell line (HMCL) was treated with different concentrations of aldosterone. (2) Confocal laser scanning microscope was used to observe the change of autophagy points after transforming eukaryotic expression vector GFP-LC3 into HMCL cells. (3) Transmission electron microscopy was used to observe the autophagic vacuoles of HMCL cells after treatment with aldosterone. Microscope and Western blotting analysis were used to observe the expression of apoptosis protein and poly(ADP-ribose) polymerase in mesangial cells under the conditions of oxidative stress (2.5×10-6 mol/L H2O2 solution) and under the presence or absence of aldosterone. Results All the three methods confirmed that high physiological dose of aldosterone (10-7 mol/L) could inhibit mesangial cell autophagy activation: (1) The autophagy marker LC3Ⅰ converting to LC3Ⅱ had a decrease of approximately 40% after 10-7 mol/L aldosterone stimulation for 12 h. (2) The numbers of autophagy point of mesangial cells induced by Earle's balanced salt solution (EBSS) and rapamycin were also reduced by 60% and 47% upon aldosterone treatment, respectively. (3) Aldosterone treatment significantly reduced starvation and rapamycin-induced vacuole formation in mesangial cells examined by transmission electron microscopy. The mesangial cells treated by aldosterone had a significantly increased apoptosis rate than the control group under oxidative stress condition with hydrogen peroxide stimulation (2.5×10-6 mol/L, P <0.05). Conclusion High physiological concentration of aldosterone shows an inhibition against the basic autophagy activation of mesangial cells and accelerates cell apoptosis under conditions of oxidative stress.

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  • 收稿日期:2015-04-01
  • 最后修改日期:2015-06-16
  • 录用日期:2015-07-07
  • 在线发布日期: 2015-08-21
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