miR-335-5P调控的TGF-β通路介导妊娠糖尿病小鼠胰岛素抵抗和胰岛β细胞分泌的实验研究

Insulin resistance and islet β cell secretion mediated by the miR-335-5P-regulated TGF-β pathway in gestational diabetes mellitus mice

目的使用miR-335-5P和转化因子-β(TGF-β)处理妊娠期糖尿病(GDM)小鼠,明确miR-335-5P调控TGF-β通路参与促进胰岛β细胞分泌和增强胰岛素抵抗机制,为治疗GDM患者开辟新道路。方法SPF级Balb/c小鼠以雌性和雄性小鼠2∶1合笼饲养,10只正常妊娠小鼠作为空白组,48只成功构建GDM模型的小鼠作为GDM组并随机分成A、B、C、D、E、F组,每组各8只小鼠。A组(模型组)、B组(模型组+溶媒)、C组(模型组+miR-335-5P模拟物)、D组(模型组+miR-335-5P抑制剂)、E组(模型组+si-TGF-β)、F组(模型组+miR-335-5P抑制剂+si-TGF-β)。药物处理后行口服葡萄糖耐量试验(OGTT)检测空腹血糖(FBG)、空腹胰岛素(FIRI),计算稳态模型胰岛素抵抗指数(IRI);进行高血糖钳夹试验测定葡萄糖输注速率(GIR),估计胰岛β细胞功能;采用RT-qPCR、蛋白质印迹分析和ELISA胰岛素释放测定胰岛β细胞中miR-335-5P、TGF-β通路关键因子mRNA水平。结果D、F组FBG、FIRI、IRI低于给药前低于C、E组(P<0.05);D、F组GIR、第一时相胰岛素、最大胰岛素高于给药前高于C、E组(P<0.05);D、F组miR-335-5P、TGF-β、c-Myc表达低于C、E组低于A、B组(P<0.05);在胰岛β细胞中检测到miR-335-5P表达时,TGF-β、c-Myc均有不同程度的表达;TGF-β在胰岛β细胞胞质内表达,且D、F组TGF-β表达高于C、E组高于A、B组(P<0.05)。结论miR-335-5P可上调GDM小鼠c-Myc表达,进一步激活TGF-β通路,增强胰岛素抵抗并抑制胰岛β细胞分泌。

Objective To investigate the mechanism by which miR-335-5P regulates the transforming factor-β(TGF-β) pathway in promoting islet β cell secretion and enhancing insulin resistance in gestational diabetes mellitus(GDM) mice. Methods Specific pathogen free grade Balb/ c mice were reared in cages at a female to male ratio of 2∶1.Ten normal pregnant mice were assigned to the blank group, and 48 mice were prepared as GDM model mice and dividedinto six groups for different drug treatments—groups A, B, C, D, E and F—with eight mice in each group. After drugtreatment, fasting glucose (FBG) and fasting insulin (FIRI) were detected by the oral glucose tolerance test (OGTT),and the insulin resistance index (IRI) was calculated. The hyperinsulinemic-euglycemic clamp was used to determine theglucose infusion rate (GIR) and to estimate pancreatic islet β cell function. The levels of miR-335-5P and key islet β cellTGF-β pathway factor mRNAs and proteins were determined by RT-qPCR, western blot and ELISA analysis. Results After drug treatment, FBG, FIRI and IRI in groups D and F were decreased and were lower than respective values of groupsC and E (P<0. 05). GIR, first-phase insulin and maximum insulin in groups D and F were increased and were higher thanrespective values of groups C and E (P<0. 05). The levels of miR-335-5P, TGF-β and c-Myc in groups D and F werelower than those of groups A, B, C and E (P<0. 05). When miR-335-5P expression was detected in islet β cells, TGF-βand c-Myc were expressed at different levels. TGF-β was expressed in the cytoplasm of islet β cells, and the expression ofTGF-β in groups D and F was higher than that in groups A, B, C and E (P<0. 05). Conclusions miR-335-5P can upregulatec-Myc expression in GDM mice, activate the TGF-β pathway, enhance insulin resistance and inhibit islet β-cellsecretion.