上海交通大学学报（医学版）

›› 2012, Vol. 32 ›› Issue (3): 288-.doi: 10.3969/j.issn.1674-8115.2012.03.011

• 论著（基础研究） • 上一篇    下一篇

醛固酮诱导MIN6细胞凋亡及作用机制研究

潘 瑜, 刘晓莉, 束金莲, 张 霞, 金惠敏   

  1. 上海交通大学 医学院附属第三人民医院肾内科, 上海 201900
  • 出版日期:2012-03-28 发布日期:2012-03-28
  • 通讯作者: 金惠敏, 电子信箱: hmjgli@yahoo.com.cn。
  • 作者简介:潘 瑜（1980—）, 女, 住院医师, 硕士；电子信箱: pannyfish@yahoo.com.cn。
  • 基金资助:

    上海市科委基金（08411962000）；上海市教委基金（10YZ35）；上海宝山区科委基金（09-E-2）

Aldosterone-induced apoptosis of MIN6 cells and its mechanism

PAN Yu, LIU Xiao-li, SHU Jin-lian, ZHANG Xia, JIN Hui-min   

  1. Department of Nephrology, the Third People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 201900, China
  • Online:2012-03-28 Published:2012-03-28
  • Supported by:

    Shanghai Science and Technology Committee Foundation, 08411962000；Shanghai Education Committee Foundation,10YZ35；Shanghai Baoshan District Science and Technology Committee Foundation, 09-E-2

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摘要：

目的 探讨醛固酮对小鼠胰岛B细胞株MIN6细胞凋亡的影响及可能的作用机制。方法 体外培养的小鼠胰岛B细胞株MIN6 分为对照组（加入无血清的DMEM培养基）、醛固酮组（加入10、100、1 000 nmol/L醛固酮进行干预）和醛固酮+拮抗剂组（以100 nmol/L醛固酮和100 nmol/L醛固酮拮抗剂螺内酯共同干预）。采用MTT法检测细胞活性；放射免疫分析法检测葡萄糖刺激的胰岛素分泌（GSIS）；流式细胞术结合FITCAnnexin V/PI荧光染色检测细胞凋亡；ELISA法检测细胞培养上清液中Caspas-3活性；Western blotting法检测凋亡相关蛋白细胞色素C(Cyt-C)、Bcl-2、Bax和磷酸化蛋白激酶C(p-Akt)的表达。结果 MIN6细胞增殖活性随醛固酮干预浓度的升高而下降，呈现浓度依赖性。在生理糖浓度（5.6 mmol/L）和高葡萄糖浓度（28 mmol/L）环境中，醛固酮组的GSIS均显著低于与对照组（P<0.01），而醛固酮+拮抗剂组GSIS显著高于醛固酮组（P<0.01）。醛固酮组细胞凋亡率显著高于对照组（P<0.01）, 而醛固酮+拮抗剂组细胞凋亡率显著低于醛固酮组（P<0.01）。与对照组比较，醛固酮组Caspase-3活性明显升高，Cyt-C表达上调，Bcl-2/Bax下降，p-Akt表达下调（均P<0.01）；而醛固酮+拮抗剂组对醛固酮组的Caspase-3活性升高及相关蛋白表达异常具有明显抑制作用。结论 醛固酮具有促进MIN6细胞凋亡的作用，其作用机制可能与Cyt-C、Bcl-2、Bax和Akt介导的线粒体信号途径有关。

关键词: 醛固酮, 胰岛B细胞, 细胞凋亡, 蛋白激酶C

Abstract:

Objective To investigate the effects of aldosterone on apoptosis of murine pancreatic islets B cell line MIN6, and explore its possible mechanism. Methods Murine pancreatic islets B cell line MIN6 cultured in vitro was divided into control group (treated with serum-free DMEM culture medium), aldosterone group (treated with 10, 100 or 1 000 nmol/L aldosterone) and aldosterone+aldosterone antagonist group (treated with 100 nmol/L aldosterone and 100 nmol/L aldactone). Cell viability was determined by MTT assay, glucose-stimulated insulin secretion (GSIS) was measured by radioimmunoassay, cell apoptosis was detected by flow cytometry in combination with FITC-Annexin V/PI fluorescein staining, Caspase-3 activity in supernatant of culture fluid was determined by ELISA, and the expression of apoptosisrelated proteins of cytochrome C(Cyt-C), Bcl-2, Bax and phosphorylated protein kinase C (p-Akt) was detected by Western blotting. Results The viability of MIN6 cells decreased with the increase of concentrations of aldosterone, with a concentration-dependent manner. Under physical glucose concentration (5.6 mmol/L) and high glucose concentration (28 mmol/L) environment, GSIS of aldosterone group was significantly lower than that of control group (P<0.01), and GSIS of aldosterone+aldosterone antagonist group was significantly higher than that of aldosterone group (P<0.01). The cell apoptosis ratio of aldosterone group was significantly higher than that of control group (P<0.01), and the cell apoptosis ratio of aldosterone+aldosterone antagonist group was significantly lower than that of aldosterone group (P<0.01). Compared with control group, the Caspase-3 activity and expression of Cyt-C were significantly higher, and Bcl-2/Bax and the expression of p-Akt were significantly lower (P<0.01 for all), while aldosterone antagonist significantly inhibited aldosterone-mediated Caspase-3 activity increase and abnormal expression of related proteins. Conclusion Aldosterone enhances apoptosis of MIN6 cells, which may be associated with Cyt-C, Bcl-2, Bax and Akt-mediated mitochondria signaling pathway.

Key words: aldosterone, pancreatic islet B cells, apoptosis, protein kinase C