GSK-3β在糖皮质激素诱导胰岛β细胞功能衰竭中的作用及分子机制研究

Steroid diabetes is the most frequent side effect of glucocorticoid treatment. The apoptosis of pancreatic β cells trigered directly by glucocorticoid is the main reason, but the underlying mechanism is remain unclear. Our previous study found that glucocorticoid induced apoptosis of INS-1 cell in dose-dependent way, and un-regulated GSK-3β level. The apoptosis could be inhibited by GSK-3 inhibitor, which indicated the key roles of GSK-3β in glucocorticoid-induced apoptosis of pancreatic β cells. In this study, we use steroid diabetes animal models and β cell line with inducible expression of GSK-3β, which have been established successfully during our previous work. First, we detect the effects of GSK-3β over expression or low expression on apoptosis and decreased insulin release induced glucocorticoid; then the underlying mechanisms of GSK-3β in this process will be analyzed by investigating mitochondrial function, Ca2+ concentration, apoptosis mediators, caspases and transfactor factors specific with β cells function, and the target molecules of GSK-3β in pancreatic β cells will be detect by immonoprecipation technology; in the latter, the signaling mechanisms involved upregulation of GSK-3β by glucocorticoid will be analyzed and inhibitors of GSK-3 which can protect pancreatic β cell also be screened.. This study investigate the roles of GSK-3β in pancreatic β cell failure from the aspect of direct injury of glucocorticoid, which will provide experimental data for unraveling the underlying mechanism of steroid diabetes and preventing and treating β cell failure in steroid diabetes.

类固醇性糖尿病是糖皮质激素临床应用中最常见的副作用。激素直接诱导胰岛β细胞衰竭是其重要原因，但相关分子机制仍不明了。我们前期发现，激素诱导胰岛β细胞凋亡伴有GSK-3β表达的升高，而抑制GSK-3活性可明显减轻激素诱导的β细胞凋亡，表明GSK-3β在激素诱导β细胞凋亡中发挥重要作用。本研究中，我们应用激素性糖尿病的动物模型和已构建成功的可诱导GSK-3β过表达的胰岛β细胞系，首先检测GSK-3β对激素诱导β细胞衰竭的影响；然后通过检测GSK-3β对线粒体、胞内钙、凋亡信号分子、caspases和β细胞特异转录因子的影响分析其作用机制，采用免疫共沉淀技术明确GSK-3β作用的靶分子；此外，拟对激素上调GSK-3β的信号机制进行分析，并筛选保护胰岛β细胞的GSK-3抑制剂。本研究从激素对β细胞直接损伤的角度探索GSK-3β在其中的作用机制，为阐明类固醇性糖尿病的发病机制和药物干预提供实验依据。

类固醇性糖尿病是糖皮质激素临床应用中最常见的副作用之一。激素直接诱导胰岛β细胞衰竭是其重要原因，但相关分子机制仍不明了。本课题研究发现地塞米松（Dexamethasone，Dex）可在体内外诱导胰岛β细胞衰竭，体外实验显示：Dex作用可明显上调INS-1细胞GSK-3β、PKC-β和c-Jun基因的表达，以GSK-3β上调较显著，同时下调AKT2和Wnt3基因表达，提示Dex诱导的INS-1凋亡可能与GSK-3β信号通路、PI3K信号通路和Wnt信号通路有关。我们选择GSK-3β信号通路为研究对象，首先利用tet-on系统成功构建了可用强力霉素（Doxycycline，Dox）诱导GSK-3β过表达的INS-1细胞系，发现GSK-3β的磷酸化水平和磷酸化位点是介导Dex诱导胰岛细胞损伤的关键，Dex可降低GSK-3β Ser9磷酸化水平，升高GSK-3βTyr216的磷酸化水平，使GSK-3β活性明显增强，进而下调Bcl-2表达、上调Bax和Capase-3表达；同时还可上调DRP-1和CREST基因表达，促进线粒体分裂，引起细胞色素C释放，使胞浆中钙浓度升高，ROS、MDA、NADPH氧化酶系统产生增多，SOD、GSH等抗氧化物质减少，促进细胞凋亡，而这些现象在GSK-3β过表达的细胞更加明显。敲降GSK-3β或应用GSK-3β抑制剂（氯化锂、SB415286）可明显减轻Dex或内质网应激诱导的胰岛β细胞凋亡。进一步我们发现Dex诱导胰岛细胞凋亡的同时，还可抑制胰岛素的合成。结果显示：Dex下调INS-1细胞胰岛素mRNA表达，同时使转录因子FoxO1由胞浆转到胞核内的数量增多，说明INS-1胞核有活性FoxO1增多，活性形式FoxO1可抑制PDX-1表达，增加PDX-1核输出，从而抑制胰岛素基因转录。而且体内实验也发现：Dex可通过调控大鼠胰腺组织GSK-3β的磷酸化水平，提高GSK-3β活性，下调胰岛细胞PI、MafA和PDX-1的表达，提示Dex抑制胰岛素合成可能与GSK-3β活性增强介导的MafA和PDX-1表达下调有关。 并且高脂血症时上述现象更加明显。但详细的机制还有待于进一步研究。因此，认为GSK-3β信号通路在介导地塞米松诱导胰岛β细胞衰竭过程中起重要作用，调控GSK-3β活性可能是临床防治类固醇糖尿病胰岛β细胞功能衰竭的潜在措施。