二甲双胍对1型糖尿病幼鼠胰岛β细胞的作用机制研究

Mechanism of action of metformin on islet β cells in type 1 diabetic rats

摘要:

目的： 探讨二甲双胍（metformin，Met）对1型糖尿病（type 1 diabetes mellitus，T1DM）幼鼠胰岛β细胞功能的影响及可能的作用机制。方法： 将健康幼鼠分为对照组（Control组）12只、诱导1型糖尿病模型组（T1DM model组）24只以及诱导1型糖尿病后Met给药组（T1DM+Met组）24只。采用葡萄糖耐量实验、胰岛素耐量实验及高葡萄糖钳夹技术检测胰岛功能；HE染色观察胰岛形态；胰岛素和Ki67免疫荧光双染法测β细胞增殖率；TUNEL法检测β细胞凋亡；Western blot检测胰腺组织中胰岛素促进因子1（pancreatic duodenal homeobox 1，PDX-1）、B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）及内质网应激相关因子的蛋白水平；RT-qPCR检测胰腺组织中炎症相关因子的mRNA水平。结果： Met可降低T1DM幼鼠空腹血糖和胰岛素水平，改善糖耐量异常和胰岛素敏感性。Met可使高葡萄糖钳夹实验中的葡萄糖输注速率（glucose infusion rate，GIR）、静脉葡萄糖刺激后1 min的胰岛素分泌量和钳夹稳态时的胰岛β细胞最大分泌能力明显增加。Met可使胰岛形态在一定程度上得到恢复。Met可通过促进PDX-1和Bcl-2蛋白水平的表达进而促进β细胞增殖，抑制其凋亡。Met能明显下调磷酸化真核起始因子2α（eukaryotic initiation factor 2 alpha，eIF2α）、活化转录因子4（activating transcription factor 4，ATF4）及C/EBP同源蛋白（C/EBP-homologous protein，CHOP）的表达（F=81.322、215.372、55.059，均P=0.000），也能明显下调核因子-κB（nuclear factor κappa-B，NF-κB）、白介素-6（interleukin 6，IL-6）及肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的mRNA水平（F=570.275、369.184、474.006，均P=0.000）。结论： Met可通过促分化、抗凋亡、缓解内质网应激压力及降低炎症反应，进而改善T1DM幼鼠胰岛β细胞的分泌功能，并能促进其增殖以及恢复胰岛形态。

Abstract:

Objective: To investigate the effect of metformin (Met) on islet β-cell function in young rats with type 1 diabetes mellitus (T1DM) and its possible mechanisms of action. Methods: Healthy young rats were recruited in this study, 12 in Control group, 24 in induced type 1 diabetes model (T1DM model) group, and 24 in Met administration group (T1DM+Met group) after induction of T1DM. Glucose tolerance test, insulin tolerance test and high glucose clamp technique were used to detect islet function. The morphology of islet was observed by hematoxylin-eosin (HE) staining. Insulin and Ki67 immunofluorescence double staining method was used to determine the proliferation rate of β cells. Apoptosis of β cells was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. Western blot was used to detect the protein levels of pancreatic duodenal homeobox 1 (PDX-1), B-cell lymphoma-2 (Bcl-2) and endoplasmic reticulum stress-related factors in pancreatic tissue. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect mRNA levels of inflammation-related factors in pancreatic tissue. Results: Met could reduce the fasting blood glucose and insulin levels of T1DM young rats, and improve the abnormal glucose tolerance and insulin sensitivity. Met significantly increased the glucose infusion rate (GIR) value, the insulin secretion 1 min after intravenous glucose stimulation, and the maximum secretion capacity of islet β cells in the clamp steady state in the high glucose clamp experiment. Met restored the islet morphology to a certain extent, promoted β cells proliferation and inhibited their apoptosis by promoting the expression of PDX-1 and Bcl-2 protein levels. Met significantly down-regulated the expression of phosphorylated eukaryotic initiation factor 2 alpha (eIF2α), eukaryotic initiation factor 4 (ATF4) and C/EBP-homologous protein (CHOP) (F=81.322, 215.372, 55.059, all P=0.000), and also significantly down-regulated the mRNA levels of nuclear factor κappa-B (NF-κB), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) (F=570.275, 369.184, 474.006, all P=0.000). Conclusion: Met can improve the secretion function of islet β cells in type 1 diabetic rats by promoting differentiation, anti-apoptosis, relieving stress of endoplasmic reticulum stress and reducing inflammation, thereby promoting its proliferation and restoring islet morphology in young rats with T1DM.