醛固酮诱导MIN6细胞凋亡及作用机制研究

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醛同酮进行干预)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.

Aldosterone-induced apoptosis of MIN6 cells and its mechanism

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.