二氮嗪干预氧化应激介导的INS-1细胞KATP通道作用机制的研究

目的研究二氮嗪(diazoxide,DIZ)干预氧化应激对INS-1细胞ATP敏感性钾通道(Adenosine triphosphate-sensitive potassium channels,KATP channels)SUR1亚基基因的表达影响。方法将INS-1细胞株分为:①氧化应激组:将50、100、200、400μmol/L H2O2分别加入各瓶细胞中培养3 h;②二氮嗪干预组:加入50μmol/L二氮嗪培养INS-1细胞30 min后,加入400μmol/L H2O2,继续培养3 h;③牛磺酸干预组:加入1 mmol/L牛磺酸培养INS-1细胞30 min后,加入400μmol/L H2O2,继续培养3 h;④空白对照组:不加二氮嗪、牛磺酸及H2O2,其他培养条件相同。MTT检测氧化应激组细胞存活率、Elisa检测葡萄糖刺激的胰岛素分泌(glucose-stimulated insulin secretion,GSIS)、Western blot分别检测3组各SUR1亚基蛋白的表达。结果 H2O2处理INS-1细胞3 h与对照组相比较后50μmol/L组细胞存活率(105.27±3.83)%稍升高(P<0.05),200μmol/L组、400μmol/L组细胞存活率分别为68.66%和51.67%均显著降低(P<0.05、P<0.01);与对照组(112.87±9.28)mU/L相比较,50μmol/L H2O2组GSIS量(118.47±10.15)mU/L升高(P<0.05);200μmol/L组(85.79±7.03)mU/L、400μmol/L组(64.59±4.53)mU/L细胞均显著降低(P<0.05,P<0.01);不同浓度H2O2培养INS-1细胞3 h后SUR1亚基蛋白的表达50μmol/L组较正常组有升高(P<0.05),100μmol/L H2O2组较正常组无统计学差异,200、400μmol/L组比对照组明显升高(P<0.05);二氮嗪干预组SUR1亚基蛋白表达比对照组显著升高(P<0.01),牛磺酸干预组与对照组比较无明显升高(P>0.05)。结论低浓度H2O2可以促进细胞增殖与胰岛素分泌,中、高浓度H2O2抑制细胞增殖与胰岛素分泌,二氮嗪抑制细胞KATP通道SUR1亚基的表达,可能是通过抑制线粒体呼吸链电子转运抑制ROS的释放,减轻细胞氧化应激的程度。

Mechanism of adenosine triphophate sensitive potassium channel in INS-1 cells mediated by diazoxide-interfered oxidative stress

Objective To study the effect of diazoxide(DIZ)-interfered oxidative stress on expression of SUR1 subunit gene in adenosine triphosphate sensitive potassium(KATP) channel of INS-I cells.Methods INS-1 cell strains were divided into oxidative stress group,DIZ interfere group,taurine interfere group,and control group.H2O2 was added into INS-1 cell strains of oxidative stress group at the concentration of 50 μmol/L,100 μmol/L,200 μmol/L,and 400 μmol/L respectively,and cultured for 3 h.DIZ was added into INS-1 cell strains of DIZ interfere group at the concentration of 50 μmol/L and incubated for 30 min,then DIZ was added at the concentration of 400 μmol/L and incubated for an additional 3 h.Taurine was added into INS-1 cell strains of taurine interfere group at the concentration of 1μmol/L and incubated for 30 min,then H2O2 was added at the concentration of 400 μmol/L and cultured for an additional 3 h.No DIZ,taurine,and H2O2 were added into INS-1 cell strains of control group.Survival rate of INS-1 cell strains,glucose simulated insulin secretion(GSIS) level,and SUR1 subunit protein expression level were measured by MTT assay,ELISA,and Western blotting,respectively,in oxidative stress group,DIZ interfere group,and taurine interfere group.Results The survival rate of INS-1 cell strains (105.27±3.83)%] was significantly higher in oxidative stress group than in control group after exposed to 50 μmol/L H2O2(P<0.05) and gradually decreased with the increased concentration of H2O2.The survival rate of INS-1 cell strains was 68.66% and 51.67%,respectively,in oxidative stress group after exposed to 200 μmol/L and 400 μmol/L of H2O2,which was significantly lower than that in control group(P<0.05).The GSIS level was higher in oxidative stress group than in control group after exposed to 50 μmol/L H2O2(118.47±10.15 mU/L vs 112.87±9.28 mU/L,P<0.05) and gradually decreased with the increased concentration of H2O2.The GSIS level was significantly lower in oxidative stress group than in control group after exposed to 200 μmol/L and 400 μmol/L of H2O2(64.59±4.53 mU/L vs 85.79±7.03 mU/L,P<0.05).The expression level of SUR1subunit protein in INS-1 cells was higher in oxidative stress group than in control group after exposed to 50 μmol/L H2O2 for 3 h and significantly higher in DIZ group than in control group after exposed to 200 μmol/L and 400 μmol/L H2O2 for 3 h(P<0.05).However,no significant difference was observed between taurine interfere group and control group(P>0.05).Conclusion DIZ inhibits the expression of SUR1 subunit gene in KATP channel of INS-I cells by suppressing the release of ROS through inhibiting the electronic transport in respiration chain of mitochrondria,thus reducing the oxidative stress of INS-I cells.