GPR40受体在阿尔茨海默症发病和治疗中的作用和机制研究

GPR40 is a member of G protein–coupled receptors (GPCR) which is lately found. It could be activated by polyunsaturated fatty acids (PUFA) and enhance the phosphorylation of cyclic AMP response element binding protein (CREB), improve the expression of brain derived neurotrophic factor (BDNF) and promote the growth of neuron. The expression of phosphorylation CREB (p-CREB) in the brains of Alzheimer's disease (AD) patients was very low and the signal pathway mediated by CREB is suppressed. The previous research work of our group has demonstrated that GPR40 receptor agonist GW9508 could significantly ameliorated cognitive performance in Aβ1-42-induced AD model mice and its mechanism may be related to that the activation of GPR40 receptor could promote the phosphorylation of CREB and significantly increase the expression of neurotropic factors including brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrohin-4 (NT-4) in mouse hippocampal neurons which contribute to neurogensis. The function could be significantly reversed by GPR40 receptor antagonist GW1100, this suggested that GPR40 is a potential drug target for the treatment and prevention of AD. Based on the previous study, in the present research, Hierarchical study was carried out using APP/PS1 transgenic mouse to study the PUFA-GPR40-CREB and PLC/IP3 pathways and clarify the role and mechanism of GPR40 on AD in pathogenesis and therapy which provide experimental evidence and ideas in search of the therapeutic drug target of AD.

GPR40是发现较晚的G蛋白偶联受体亚型，能被多不饱和脂肪酸激活，促进环磷腺苷效应元件结合蛋白（CREB)磷酸化,提高BDNF表达,促进神经生长。在阿尔茨海默症（AD）患者脑内，磷酸化的CREB（p-CREB）水平较低，由CREB介导的信号通路被抑制。我们前期工作显示：GPR40受体激动剂GW9508可以改善Aβ1-42 AD模型小鼠的记忆认知障碍，其机制可能与提高海马区p-CREB和神经营养因子（BDNF、NGF、NT-3、NT-4）的表达相关，该作用可被GPR40受体拮抗剂GW1100所阻断。这提示GPR40受体可能作为AD治疗新靶点。本研究在此基础上围绕GPR40受体，采用APP/PS1双转基因AD小鼠,从体内外以及PUFA-GPR40-CREB和PLC/IP3两条通路，开展多层次的系统研究，为阐明GPR40在AD发病和治疗中的作用和机制，寻找AD治疗药物新靶点提供实验依据和思路。

本项目采用CHO 细胞构建GPR40稳转株及GPR40受体激动剂筛选模型并用GW9508进行了模型确证。采用APP/PS1双转基因B6C3-Tg阿尔茨海默症（AD）模型小鼠，设置正常对照组（同窝出生的转基因阴性小鼠）、AD模型组、阳性药（多奈哌齐）组、GPR40受体激动剂GW9508和TAK-875低、中、高剂量组、GPR40 受体拮抗剂 GW1100+GPR40受体激动剂高剂量组。通过 Morris 水迷宫等经典行为学实验考察了GW9508和TAK-875对AD小鼠认知功能障碍的改善作用及GPR40 拮抗剂GW1100对该作用的影响。行为学实验后，分离各组小鼠海马和皮层脑组织，采用Western Blot检测各脑区CREB、p-CREB、PLC、p-PLC、PKC表达，ELISA检测神经营养因子BDNF、NGF、NT-3、NT-4含量，高内涵技术和免疫荧光检测促神经元生长作用，研究阐明了GPR40受体激动剂经PUFA-GPR40-PKA-CREB和PLC//IP3信号通路改善AD的分子机制。采用Aβ1-42诱导原代培养大鼠皮层神经元AD损伤，Western blot检测CREB、p-CREB、JNK、p-JNK表达，ELISA检测Aβ1-42、IL-6、IL-1β、TNF-α、BDNF、NGF、NT-3、NT-4含量，研究阐明了GPR40受体激动剂经JNK信号通路减少神经元Aβ沉积的作用机制。相关成果在Neropharmacology、International Immunopharmacology、Behavioural Brain Research、Journal of Drug Targeting等SCI行业经典杂志发表高水平论文11篇，阐明了GPR40受体与AD发病和治疗的相关性及GPR40 受体激动剂改善AD的作用与机制，为以GPR40为靶点的AD治疗药物研发提供了实验依据。