Objectives: The β-cell dedifferentiation is one of the critical mechanisms in diabetic β-cell loss. Long-term activation of N-methyl-D-aspartate (NMDA) receptors plays an essential role in the development of diabetes, but the underlying mechanisms have not been fully elucidated. This study aims to investigate the effect of prolonged activation of NMDA receptors on islet β-cell dedifferentiation.
Methods: Male C57BL/6 mice were randomly divided into a normal control group (control group) and an NMDA group. The mice in the NMDA group were intraperitoneally injected with NMDA (8 mg/kg body weight) and those in the control group were injected with the same volume of saline every day for 6 months. At the end of the 6 th month, glucose tolerance and enzyme linked immunosorbent assay (ELISA) were used to detect the function of islets, and pancreatic tissues were taken for immunofluorescence staining to detect the expressions of insulin, glucagon, and proliferating cell nuclear antigen (PCNA). Real-time PCR was used to detect the mRNA expression of pancreatic β cells, α cells, and islet progenitor cell markers.The primary islets were treated with NMDA to observe the effect of NMDA on the dedifferentiation of β cells. The nuclear factor kappa-B (NF-κB) inhibitor BAY 11-7082 was used at the cellular level via detecting insulin secretion and the expression of endocrine cell markers.
Results: Compared with the control group, the mice in the NMDA group had higher blood glucose levels at each time point after glucose injection, and the area under the glucose tolerance curve was significantly increased ( P <0.05). The serum insulin content and insulin stimulatory index of the mice in the NMDA group were significantly lower than those in the control group at 30 min after glucose injection (both P <0.05). The double immunofluorescence staining for insulin and glucagon showed that the number of insulin-positive β cells in the pancreatic tissues of mice was significantly decreased after intraperitoneal injection of NMDA in mice for 6 months, while the number of glucagon-positive α cells was significantly increased. Real-time PCR results showed that β-cell markers ( Insulin , Pdx1 , Neurod1 , and Mafa ) were significantly down-regulated in mouse pancreatic tissues after intraperitoneal injection of NMDA for 6 months, while pancreatic progenitor cell markers ( Neurog3 , Gata6 , Hnf4a , Notch1, and Hes1 ) were significantly down-regulated; α-cell markers ( Glucagon , Arx , Irx2 , Mafb , Pou6f2 , Fev , Kcnj3, and Sv2b ) were significantly up-regulated. NMDA treatment of mouse primary islets for 48 h cause significant down-regulation of β-cell marker gene expression ( P <0.05 or P <0.01), accompanied by significant up-regulation of pancreatic progenitor cell markers and α-cell markers ( P <0.05, P <0.01 or P <0.001). The NF-κB inhibitor BAY 11-7082 significantly blocked the down-regulation of β-cell marker expression (all P <0.05) and the up-regulation of α-cell and pancreatic progenitor cell marker after NMDA treatment of islets for 48 h ( P <0.05 or P <0.01).
Conclusions: Prolonged activation of NMDA receptors induces islet β-cell dedifferentiation via regulating the NF-κB pathway.
目的 : β细胞去分化是糖尿病β细胞丢失的关键病理机制之一。N-甲基-D-天冬氨酸(N-methyl-D-aspartate，NMDA)受体长时间激活在糖尿病发生和发展中扮演着重要角色，但其机制尚未完全阐明。本研究旨在探讨NMDA受体长时间激活对胰岛β细胞去分化的影响及其作用机制。 方法 : 将雄性C57BL/6小鼠随机分为正常对照组(Control组)和NMDA组，每天于小鼠腹腔注射NMDA(8 mg/kg体重)或同等体积生理盐水，连续注射6个月。第6个月末进行葡萄糖耐量试验及使用酸联免疫吸附试验(enzyme linked immunosorbent assay，ELISA)检测血清中胰岛素分泌情况，并取胰腺组织进行免疫荧光染色检测胰岛素(insulin)、胰高血糖素(glucagon)和增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)表达情况；采用real-time PCR检测胰岛β细胞、α细胞及胰岛祖细胞标志物mRNA表达。用NMDA处理原代胰岛观察NMDA对胰岛β细胞去分化的影响，再在细胞水平使用核因子kappa-B(nuclear factor kappa-B，NF-κB)抑制剂BAY 11-7082，通过检测胰岛素分泌情况以及内分泌细胞标志物的表达探讨NMDA诱导胰岛β细胞去分化的机制。 结果 : 与对照组相比，NMDA处理组小鼠注射葡萄糖后各个时间点的血糖值均增高，且糖耐量曲线下面积显著增加(均 P <0.05)。NMDA组小鼠在葡萄糖注射后30 min血清中insulin含量以及胰岛素刺激指数显著低于对照组(均 P <0.05)。Insulin与glucagon免疫双荧光染色结果显示：小鼠腹腔注射NMDA 6个月后，小鼠胰腺组织中insulin阳性的β细胞数量明显减少，而glucagon阳性的α细胞数量明显增加。Real-time PCR结果显示，腹腔注射NMDA 6个月后，小鼠胰腺组织中β细胞标志物( Insulin 、 Pdx1 、 Neurod1 和 Mafa )显著下调，而胰腺祖细胞标志物( Neurog3 、 Gata6 、 Hnf4a 、 Notch1 和 Hes1 )和α细胞标志物( Glucagon 、 Arx 、 Irx2 、 Mafb 、 Pou6f2 、 Fev 、 Kcnj3 和 Sv2b )显著上调。NMDA处理原代小鼠胰岛48 h可以诱导β细胞标志基因表达显著下调( P <0.05或 P <0.01)，伴随着胰腺祖细胞标记物和α细胞标志物的显著上调( P <0.05， P <0.05或 P <0.001)。NF-κB抑制剂BAY 11-7082能显著减轻NMDA处理胰岛48 h后β细胞标志物表达的下降(均 P <0.05)和胰岛α细胞和胰腺祖细胞标志物表达的上调( P <0.05或 P <0.01)。 结论 : NMDA受体长时间激活诱导胰岛β细胞去分化，其机制有赖于NF-κB的介导。.
Keywords: N-methyl-D-aspartate; cell differentiation; islet β cell; nuclear factor kappa-B.