氯化锂对地塞米松诱导的大鼠胰岛β细胞凋亡的影响及其机制

Effect of lithium chloride on apoptosis of islet β cells in rats induced by dexamethasone and its mechanism

目的:建立地塞米松(Dex)诱导的大鼠胰岛INS-1细胞凋亡模型,探讨氯化锂(LiCl)对Dex诱导的胰岛β细胞凋亡的影响及其可能机制。方法:将INS-1细胞分为对照组、0.1μmol·L^-1 Dex组和LiCl+0.1μmol·L^-1 Dex组。TUNEL染色和AnnexinⅤ/PI染色法检测各组INS-1细胞凋亡率,Real-time PCR法检测各组INS-1细胞中超氧化物歧化酶(SOD)、诱导型-氧化氮合酶(iNOS)、NADPH氧化酶4(Nox4)、NADPH oxidase(p47phox)和糖原合成酶激酶3β(GSK-3β)mRNA表达水平,Western blotting法检测各组INS-1细胞中GSK-3β、p-GSK-3、SOD、iNOS和Nox4蛋白表达水平,GENMED试剂盒检测各组NIS-1细胞中活性氧(ROS)水平,Griess法检测各组INS-1细胞中一氧化氮(NO)水平。结果:与对照组比较,0.1μmol·L^-1 Dex组INS-1细胞凋亡率升高(P<0.05),SOD mRNA和p-GSK-3β蛋白表达水平降低(P<0.05),细胞中Nox4、p47phox、iNOS mRNA表达水平升高(P<0.05),细胞中ROS和NO水平升高(P<0.05);与0.1μmol·L^-1 Dex组比较,LiCl+0.1μmol·L^-1 Dex组INS-1细胞凋亡率降低(P<0.05),SOD mRNA和p-GSK-3β蛋白表达水平升高(P<0.05),细胞中Nox4、p47phox和iNOS mRNA表达水平降低(P<0.05或P<0.01),细胞中ROS和NO水平降低(P<0.05)。结论:Dex可诱导大鼠胰岛β细胞凋亡,LiCl可通过抑制GSK-3β活性减轻Dex诱导的胰岛β细胞凋亡。

Objective:To construct the apoptosis model of islet INS-1 cells of the rats induced by dexamethasone(Dex),and to investigate the effect of lithium chloride(LiCl)on the apoptosis of the isletβcells induced by Dex and its possible mechanism.Methods:The INS-1 cells were divided into control group,0.1μmol·L^-1 Dex group and LiCl+0.1μmol·L^-1 Dex group.TUNEL staining and AnnexinⅤ/PI staining methods were used to detect the apoptotic rates of the INS-1 cells in various groups;Real-time PCR method was used to detect the expression levels of superoxide dismutase(SOD),inducible-nitric oxidesynthase(iNOS),NADPH oxidase 4(Nox4),NADPH oxidase(p47phox),and glycogen-synthase kinase-3β(GSK3β)mRNA in the INS-1 cells in various groups;Western blotting method was used to detect the expression levels of GSK-3β,p-GSK-3β,SOD,iNOS and Nox4 proteins in the INS-1 cells in various groups;GENMED Kit was used to detect the levels of reactive oxygen species(ROS)in the INS-1 cells in various groups,and Griess method was used to detect the levels of nitric oxide(NO)in the INS-1 cells in various groups.Results:Compared with control group,the apoptotic rate of INS-1 cells in 0.1μmol·L^-1 Dex group was significantly increased(P<0.05),the expression levels of SOD mRNA and p-GSK-3βproteins were decreased(P<0.05),the expression levels of Nox4,p47phox,iNOS mRNA were increased(P<0.05),and the expression levels of ROS and NO in the INS-1 cells were significantly increased(P<0.05);compared with 0.1μmol·L^-1 Dex group,the apoptotic rate of INS-1 cells in LiCl+0.1μmol·L^-1 Dex group was significantly decreased(P<0.05),the expression levels of SOD mRNA and p-GSK-3βprotein were increased(P<0.05),the expression levels of Nox4,p47phox and iNOS mRNA were decreased(P<0.05 or P<0.01),and the levels of ROS and NO were decreased(P<0.05).Conclusion:Dex can induce the apoptosis of the isletβcells of the rats.LiCl can attenuate the apoptosis of dexamethasone-induced isletβcells by inhibiting the activity of GSK-3β.