瘦素调节NSCs/NPCs分化平衡促进脑损伤后神经再生修复的作用及机制研究

Neural stem cells/neural progenitor cells (NSCs/NPCs) are a group of immature cells that are self-renewing and multipotent. After brain injury, sufficient number of actively proliferating stem cells/progenitors and proper amount of neurons /glia are vitally required for neural regeneration and repair, which is similar with neural development. Elements that can help to maintain the stemness of NSCs/NPCs or induce the target differentiation of NSCs/NPCs will contribute to the neuroregeneration. .Leptin is one of the protein hormones that plays crucial role in regulating energy balance. It has also been considered as a high potential medicine for the treatment of brain injury due to its neuroprotective effects. Our previous work showed that the survival and proliferation of NSCs/NPCs in vitro were enhanced by leptin and the intrinsic NSCs/NPCs in subventricular zone of rats with hypoxic-ischemic brain injury were also activated after adaption of leptin. This suggested that leptin might work on NSCs/NPCs biological behaviors after brain injury somehow and make some contribution to neuroregeneration. However, after brain injury, the blood supply was reduced and inflammatory cytokines were recruited around the ischemic foci, whether the leptin will affect the fate determination of NSCs/NPCs, in other words, will leptin affect the decision of NSCs/NPCs to either proliferate or differentiate, to differentiate into neurons or glia? How does leptin work? All these questions still remain to be seen. .Some existed data and our previous work showed that the classic downstream signaling pathways of leptin, such as JAK-STAT3, MAPK/ERK, PI3K/AKT and AMPK, are involved in the regulation of neural regeneration as well as the energy balance. In addition, metabolic plasticity plays important roles in maintaining stemness and lineage specification of NSCs/NPCs. Therefore, we hypothesized that leptin might affect the proliferation and differentiation of NSCs/NPCs via both the classic signaling pathways and the regulating of metabolic plasticity, which including function of mitochondrion and the metabolism of glucose and lipid acid, and eventually enhance the neural regeneration. .In order to prove our hypothesis, we intend to set up the oxygen-glucose derived (OGD) models on both primary cultured NSCs/NPCs and organotypic brain slices, the middle cerebral artery occlusion (MCAO) model on adult SD rats. Leptin will be used in different time points combine with/without extrinsic CM-DiI pre-labeled NSCs/NPCs transplantation. BrdU will be applied for the intrinsic NSCs/NPCs labeling. Then the number and distribution of NSCs/NPCs and the differentiated neurons/glia will be investigated to illustrate the effect of leptin on the survival, proliferation and differentiation of NSCs/NPCs in the injured condition. Single cell sequencing will be used to identify the stage specific transcription factors that related to the different stages of NSCs/NPCs proliferation or differentiation induced by leptin. Meanwhile, the morphology and function of mitochondrion, the metabolic alteration of glucose and lipid acid will be observed and their influence on NSCs/NPCs biological behavior will be analyzed. The interaction of intrinsic and extrinsic NSCs/NPCs, the integration of NSCs/NPCs with local neurons will be also detected from both morphological and functional aspects. At last, the movement scores of MCAO rats will be measured to demonstrate the promotion of functional recovery. Our results will benefit the development of new therapeutic strategy for brain injury that based on leptin and NSCs/NPCs biological behaviors.

神经干/前体细胞（NSCs/NPCs）分化平衡（即细胞选择增殖或者分化、向哪种类型细胞分化），对脑损伤后神经再生修复至关重要。已证实机体代谢状态影响神经再生，瘦素下游信号通路也不同程度参与神经再生调控。那么脑损伤后，瘦素作为代谢调节重要激素将如何调控NSCs/NPCs分化平衡？还需探索。基于文献和课题组前期研究，我们推测：瘦素可能通过差异性激活下游信号及调节损伤组织代谢状态，完成对NSCs/NPCs分化平衡调控，最终促进神经再生修复。本项目拟构建细胞/组织氧糖剥夺及大鼠脑缺血缺氧模型，以形态学、分子生物学、行为学及电生理检测方法，分别在细胞、组织及动物水平观察瘦素对NSCs/NPCs分化平衡及大鼠神经功能恢复的影响，结合局部细胞线粒体形态/功能、物质与能量代谢水平，阐明瘦素促进脑损伤后神经再生修复的作用及细胞分子机制，为开发基于瘦素和NSCs/NPCs分化平衡的脑损伤治疗新策略提供证据。

神经干/前体细胞（NSCs/NPCs）是神经系统的原始细胞，具有增殖和多向分化能力，为脑损伤后神经功能修复带来了希望。寻求促进NSCs/NPCs存活、增殖以及精确调控其分化方向的策略，一直是神经再生与损伤修复研究的重点。瘦素（Leptin）是由脂肪细胞分泌的蛋白类激素，不仅参与代谢调控，近年来其神经保护作用也备受关注，有望成为极具潜力的脑损伤治疗用药。本项目在前期探索的基础上，提出“瘦素可能通过差异性激活下游信号及调节损伤组织代谢状态，完成对NSCs/NPCs分化平衡调控，最终促进神经再生修复”的科学假说。项目通过构建体外细胞模型、发育期内源性高Leptin持续暴露以及脑损伤动物模型，对Leptin参与调控NSCs/NPCs分化平衡的作用及机制进行探索。结果证实①Leptin在体内、外条件下促进NSCs/NPCs存活及向神经元方向分化；PI3K/Akt、JAK2/STAT3信号通路以及Ca2+内流/钙通道激活共同参与Leptin对NSCs/NPCs分化平衡的调控；②孕期高脂饮食导致持续内源性高Leptin水平，减弱了子代海马NSCs/NPCs增殖、降低其干性，加速消耗、促进早熟，影响子代出生后早期海马发育进程；③星形胶质-小胶质细胞niche影响NSCs/NPCs增殖分化行为，Leptin通过改变胶质细胞niche影响NSCs/NPCs分化平衡，BMP4参与其调控作用；④外源性补充Leptin可减轻脑出血损伤动物的损伤面积，促进神经功能恢复，同时抑制内源性NSCs/NPCs所处niche中炎症及氧化应激水平，促进其增殖及向神经元方向分化。上述结果综合阐明了Leptin对脑损伤后NSCs/NPCs分化平衡及神经功能修复的影响及可能机制，有助于开发基于Leptin的脑损伤修复治疗新策略，也为寻找通过调节NSCs/NPCs分化平衡促进神经再生修复的有效途径提供了新的思路。