基于lncRNA及NMDA受体调控突触可塑性探讨叶酸影响脑梗死后认知功能的机制

Folic acid can impove the cognitive function following ischemic stroke. The effect of this intervention is specially obvious in some countries without mandatory folic acid food fortification. In addition, synaptic plasticity is the neurobiological basis of learning and memory, and both lncRNAs and NMDA receptors are critically involved in synaptic plasticity and cognition. Based on the analysis of the relationship between folic acid and homocysteine metabolism and the early screening experiment of lncRNA-mRNA expression profile in folate deficient neurons, we speculate that synaptic plasticity, which is mediated by both lncRNA and NMDA receptors, may play an important role in folic acid-regulating cognition. In order to verify this hypothesis, primary cultured neurons under oxygen glucose deprivation (OGD) condition,the artery occlusion model in rats and group intervention will be conducted to build the association among folic acid, lncRNA/NMDA receptors and synaptic plasticity (cognitive function). The roles of NMDA receptor subunits and lncRNAs which have been screened out in our previous studies will be clarified in the synaptic plasticity affected by folic acid intervention. Furthermore, the possible mechanisms which lncRNAs regulate the expression levels of candidate synaptic genes Nrxn1 and Shank3 will be explored by RNA pull-down technique and mass spectrometry analysis. The study will further enrich the understanding of the pathological mechanism of cerebral infarction, and provide theoretical support and experimental evidence for the prevention and treatment of folic acid on cognitive dysfunction after cerebral infarction.

叶酸可显著改善脑梗死后的认知功能，在没有实施食品叶酸强化的国家，效果尤为明显。海马突触可塑性是学习和记忆的基础，lncRNA及NMDA受体可调控突触可塑性进而影响认知。本研究在叶酸与同型半胱氨酸代谢关系分析及叶酸缺乏神经元lncRNA-mRNA表达谱筛选的前期实验基础上，提出以下假说：特异lncRNA及NMDA受体介导的突触可塑性可能在叶酸改善梗死后认知功能中发挥重要作用。拟从原代神经元氧糖剥夺模型、大鼠中动脉栓塞模型及人群干预三个方面，通过lncRNA和NMDA受体各亚基的表达、定位检测及siRNA干扰和过表达功能分析，明确lncRNA分子、NMDA受体亚基及二者协同作用在叶酸调控突触可塑性中的地位；采用RNA pull-down及质谱分析进一步研究lncRNA分子与候选突触相关基因Nrxn1和Shank3之间可能的网络调控关系。研究将为叶酸对脑梗死后认知损伤的防治作用提供实验证据。

脑梗后认知障碍是卒中常见的并发症，不仅严重影响患者生活质量，而且显著降低卒中患者生存时间，给家庭及社会带来沉重负担。已有研究表明，lncRNA分子广泛参与各种人类神经系统疾病；NMDA 受体是影响学习记忆的关键物质。叶酸是否通过调控lncRNA和NMDAR表达并改善脑梗死后的认知功能，目前国内外尚无相关报道。本研究采用原代神经元氧糖剥夺模型和大鼠局灶性脑梗塞MCAO模型进行叶酸干预，结果显示：叶酸能有效改善大鼠脑缺血后的学习记忆，减少神经元损伤和凋亡，提高了突触相关蛋白的表达，部分修复了脑梗后突触结构的破坏。NMDA和叶酸联合干预及NMDAR siRNA敲减实验均表明，NMDAR的表达水平在叶酸改善脑梗后神经损伤作用中发挥重要作用。同时通过RT-qPCR及lncRNA siRNA实验发现，叶酸可通过改变神经细胞中突触相关LncRNA分子的表达从而改善缺糖缺氧引起的细胞损伤；人群研究进一步明确了叶酸改善脑梗患者认知功能的作用；同时发现血清叶酸、NMDAR2A和NMDAR2B、某些LncRNA（如H19和MEG3）与脑梗后不同认知域有关。因此叶酸对缺血性损伤后认知功能的影响及其潜在机制可能是通过改善突触功能障碍，同时上调已知突触可塑性标志物、下调NMDARs的表达及改变某些特定LncRNA表达有关。本研究建立了叶酸—lncRNA、NMDAR—神经保护作用之间的关联关系，找到它们之间关联的直接证据。阐明叶酸促进脑梗死患者神经修复和改善认知功能的可能机制，为叶酸对脑梗死的防治作用提供理论依据和实验支持。可见在我国居民叶酸普遍缺乏、脑梗高发的情况下，开展叶酸防治脑梗死的研究具有重要的现实意义。