APP17肽对糖尿病KK小鼠海马神经元胰岛素信号转导途径的影响

Effects of Amyloid Precursor Protein 17 Peptide on the Hippocampal Neuron-insulin Signaling Transduction Pathway of Diabetic Kkay Mice

目的观察APP17肽对2型糖尿病KKAy小鼠(以下简称KK小鼠)大脑海马神经元胰岛素信号转导蛋白表达的影响。方法根据血糖水平将KK小鼠分为正常对照组(C组)、糖尿病组(DM组)和APP17肽治疗组(DM+APP17P组)。治疗组给予皮下注射APP17肽,每周3次,每次8μg/kg,共12周。处死前尾静脉取血测定糖化血红蛋白、血糖含量;将3组小鼠处死,心脏取血测血浆胰岛素;冰浴中快速分离海马组织,匀浆后,用于免疫沉淀并蛋白印迹检测,部分快速灌注固定做免疫组化染色和Tunel细胞染色。结果与C组相比,DM组与DM+APP17P组的血糖、糖化血红蛋白、胰岛素水平显著升高(P<0.05),但是DM组与DM+APP17P组之间无显著差异;DM组海马神经元胰岛素受体(InsR),胰岛素受体底物-1(IRS-1)的表达低于C组和DM+APP17 P(P<0.05);CREB、pCREB、Bc l-2的表达3组之间差异无统计学意义。Tunel细胞染色3组小鼠海马均未发现Tunel染色阳性细胞。结论2型糖尿病KK小鼠存在海马胰岛素信号转导通路的异常,主要表现为P13-K上游起始途径的异常。这种异常可通过神经元一定程度上的自我调节而改善,并不导致细胞凋亡状态;APP17肽作为神经营养因子能激活海马神经元存活信号转导通路上游成分,从而使存活信号转导通路恢复正常。

Objective Amyloid precursor protein（APP） is one in which neurotrophic and neurotoxic functions occur on the same chain. The APP319 -335 segment （APP17 peptide ） can enhance neuritic growth . Solube APP17 peptide exerts its effect through binding to specific binding sites on the cell surface. The present work is to investigate the effect of APP17 peptide on the expressions of insulin signaling transduction protein of T2 diabetic KKAy mice hippocampal neurons. Methods KK mice were divided into three groups ： a normal control group（ C, n = 10）, a diabetic group （ DM, n = 10） and an APP17mer peptide-protected diabetic group（ DM ＋ APP17P, n = 10）. Blood glucose in a tail-vein sample was determined by a glucose analyzer. A value 〉 15 mmol/L was accepted as a diabetic model. APP17mer peptide was synthesized by solid phase method and purified by HPLC . Mice in the APP17 ＋ DM group were injected peptide subcutaneously at the dose of 8 μg/kg three times a week for 12 weeks. After twelve weeks, all the mice were killed and peripheral blood were obtained to assess the levels of HbA1c, plasma insulin and blood glucose. Homogenates were prepared from mice hippocampal tissue. Immunoprecipitation, Western-blot and immunohistochemistry were used to analyze the proteins related to insulin signaling transduction pathway. Tunnel assay was used to detect the apoptotic cell in hippocampus of KK mice. Immunohistochemical assay was performed with insulin receptor（ InsR）, insulin receptor substrate-1 （ IRS-1 ） and Bcl-2. Results The results of InsR and IRS- 1 immunohistochemical staining showed that the hippocampus of C group and DM ＋ APP17P contained darkly stained InsR and IRS-1 positive cells. In contrast positive reacting cells in the hippocampus of DM group mice were pale , and were visibly fewer than in those C group and APP17P ＋ DM group mice. The number of positively stained hippocampal cells for Bcl-2 was similar in three groups. The expressions of CREB, phosphorylated CREB（p-CREB） and Bcl-2 showed no significant difference among the three groups. No positive Tunel cell was found in all groups. Conclusion Abnormal signal transduction pathway is present in hippocampal neuron of kk mice. The abnormality may not lead to apoptosis due to auto-regulation of neurons to a certain extent. This abnormal insulin signaling transduction pathway can be improved by APP17 peptide.