在糖尿病脑病中mTOR/IKK/NF-κB通路参与BDNF介导的突触可塑性调控及木犀草素的保护作用

In db/db mouse model of diabetes mellitus (DM), mTOR/IKK/NF-κB pathway could modulate the synaptic plasticity, which is mediated by the brain-derived neurotrophic factor (BDNF), with the underlying mechanism still unrevealed. In our previous study, luteolin served to downregulate the p-mTOR expression and mitigate injury to synaptic plasticity in the hippocampal in db/db mouse model of DM, with the cognitive functionality significantly ameliorated, coupled with upregulation of IκB-α, downregulations of NF-κB and pro-inflammatory cytokines and upregulation of BDNF. We speculated that mTOR could modulate IKK/IκB/NF-κB pathway and promote the release of pro-inflammatory cytokines, contributing to the impairment of synaptic plasticity. This project is focused on the modulation of IKK/IκB/NF-κB by mTOR and aimed to investigate the expressions of mTOR, NF-κB and pro-inflammatory cytokines on the animal and cellular levels, and assess the morphological and ultrastructural changes of hippocampal neurons, expression variations of BDNF and synapse-related proteins as well as fluctuations of long-term potentiation (LTP). This project will benefit the further elucidation of the mechanisms underlying the BDNF-mediated modulation of synaptic plasticity by mTOR/IKK/NF-κB pathway in diabetic encephalopathy and the neuroprotection by luteolin herein and ultimately the provision of novel strategies for the prophylaxis and therapeutics of diabetic encephalopathy.

mTOR/IKK/NF-κB通路在糖尿病脑病中通过BDNF介导可以调控突触可塑性，然而机制不明。我们先前研究发现：木犀草素可在db/db小鼠糖尿病脑病模型中下调海马p-mTOR并减轻海马突触可塑性损伤，改善db/db小鼠认知功能；也发现IκB-α表达上调、NF-κB和促炎症因子表达下调、BDNF表达上调。我们推测mTOR在糖尿病脑病中调控IKK/IκB/NF-κB信号通路，增加促炎症因子释放，降低BDNF表达，继而损伤突触可塑性。本课题以mTOR调控IKK/IκB/NF-κB信号通路为切入点，在整体及细胞水平上检测mTOR、NF-κB通路关键信号分子及促炎症因子的表达；观察海马神经元在形态学及突触超微结构的变化，检测BDNF、突触相关蛋白以及LTP变化；阐明在糖尿病脑病中mTOR/IKK/NF-κB通路参与BDNF介导的突触可塑性调控及木犀草素的神经保护作用，为该疾病的防治提供新思路。

糖尿病脑病是一种严重的糖尿病并发症，其特征在于海马神经发炎和异常突触发生，导致认知能力下降。哺乳动物雷帕霉素靶蛋白（mTOR）与认知障碍有关，核因子-κB（NF-κB）是促炎细胞因子的转录因子。尽管mTOR与神经炎症过程有关，但在糖尿病脑病中该蛋白对NF-κB信号通路的作用尚不清楚。本研究使用糖尿病脑病的体内和体外模型，验证mTOR通过调控NF-κB信号通路，调节海马神经元的神经炎症和突触可塑性；并且在动物实验中，以木犀草素给药治疗，发现木犀草素抑制海马神经元中的mTOR/NF-κB信号通路，调控下游蛋白tau、BDNF、HDAC2的表达，从而逆转糖尿病脑病所致的认知损伤。. 本项目细胞实验以高糖诱导的小鼠海马神经元HT-22细胞作为体外细胞模型。通过分别抑制mTOR或NF-κB，探讨了mTOR /NF-κB信号转导对促炎细胞因子和突触蛋白的影响。结果发现，高糖显著增加HT-22细胞中p-mTOR的表达；雷帕霉素或shmTOR抑制mTOR可以显著抑制高糖诱导的NF-κB及其调节剂IKKβ和IκBα的活化，表明mTOR是NF-κB的上游调节剂；在高糖条件下，PDTC和shNF-κB抑制NF-κB会降低HT-22细胞促炎细胞因子（IL-6，IL-1β和TNF-α）的表达，并增加脑源性神经营养因子（BDNF）和突触蛋白（SYN和PSD-95）的表达。. 本项目动物实验通过高脂饮食联合链脲佐菌素腹腔注射诱导糖尿病脑病小鼠模型，分别给予小鼠雷帕霉素（mTOR抑制剂），PDTC（NF-κB抑制剂），LMTX（tau聚集抑制剂），以及木犀草素低、高剂量给药。研究发现，在糖尿病脑病状态下，小鼠海马内mTOR被过度激活，激活的mTOR可通过调控IKKβ/IκBα/NF-κB信号通路，促进海马内促炎症因子的基因转录；激活NF-κB信号通路还可以使tau激活，进而下调BDNF的表达；神经元中激活的NF-κB会导致HDAC2表达增加，从而抑制突触相关蛋白（SYN、PSD-95）的表达，造成突触可塑性损伤，导致认知缺陷。木犀草素可以通过抑制神经元中mTOR/NF-κB信号通路，逆转以上过程，发挥防治糖尿病脑病的作用。