血管紧张素Ⅱ经线粒体途径介导RIN-m细胞凋亡的实验研究

Apoptosis of RIN-m cells mediated by angiotensin Ⅱ by mitochondrial pathway

目的探讨血管紧张素Ⅱ(AngⅡ)和氯沙坦对胰岛β细胞的作用及相关分子机制。方法常规培养大鼠胰岛素瘤RIN-m细胞,分为3组:空白对照组(RPMI1640培养基常规培养)、AngⅡ组(终浓度100nmol/LAngⅡ处理)、氯沙坦预处理组(终浓度1μmol/L氯沙坦作用15min后再添加终浓度100nmol/L的AngⅡ)。按照前述分组处理完毕后继续孵育48h。采用Annexin V-FITC/PI双染法检测细胞凋亡率,RT-PCR和Western blotting检测Bcl-2、Bax的mRNA和蛋白表达,分光光度法检测Caspase3和Caspase9活性。结果AngⅡ组细胞凋亡率明显高于空白对照组(P<0.001)和氯沙坦预处理组(P<0.001),后两组间比较无显著差异(P>0.05)。与空白对照组及氯沙坦预处理组比较,AngⅡ组Bcl-2mRNA和蛋白表达显著降低(P<0.001),BaxmRNA和蛋白表达显著增加(P<0.001)。氯沙坦预处理组Bcl-2、Bax的mRNA和蛋白表达与空白对照组比较无显著差异(P>0.05)。AngⅡ组Caspase3和Caspase9的活性显著高于空白对照组(P<0.05)及氯沙坦预处理组(P<0.05),后两组间比较无显著差异(P>0.05)。结论AngⅡ可能通过线粒体途径诱导胰岛β细胞凋亡,氯沙坦预处理可部分逆转AngⅡ的这种效应,从而对β细胞发挥保护作用。

Objective To investigate the effect of angiotensin Ⅱ（AngⅡ） and losartan on β cells,and its related molecular mechanisms.Methods Normally cultured RIN-m cells were divided into three groups：control group（cultured with RPMI 1640 medium）,AngⅡ group（treated with 100 nmol/L AngⅡ）,losartan group（treated with 1 μmol/L losartan 15min before addition of 100nmol/L AngⅡ）.After incubation for another 48h,the apoptosis rate of RIN-m cells was quantified by flow cytometry（FCM） with Annexin-V FITC/PI dual staining.The expressions of Bcl-2 and Bax mRNA and protein were determined by RT-PCR and Western blotting,and the activities of Caspase 3 and Caspase 9 were detected by spectrophotometry.Results The apoptosis rate of RIN-m cells was significantly higher in AngⅡgroup than in both control and losartan group（P〈0.001）,and no significant difference existed between the latter two groups（P〉0.05）.In comparison to the control group,the expressions of Bcl-2 mRNA and protein significantly declined,while of Bax mRNA and protein increased obviously in AngⅡ group（P〈0.001）.No significant difference existed on the expressions of Bcl-2 and Bax mRNA and protein between losartan and control group（P〉0.05）,but the expressions of Bcl-2 mRNA and protein were significantly higher（P〈0.001）,and that of Bax mRNA and protein was obviously lower（P〈0.001） in losartan group than in AngⅡ group.The activities of Caspase 3 and Caspase 9 were significantly higher in AngⅡ group than in both control and losartan group（P〈0.05）,while no significant difference was found between the latter two groups（P〉0.05）.Conclusion AngⅡ may induce the apoptosis of β cells through mitochondrial pathway,and pre-intervention with losartan,which partly reverses the effect of AngⅡ,may play a protective effect on β cells.