基于Ubiquilin-1调控神经干细胞增殖和分化探讨淫羊藿苷对新生小鼠缺氧缺血性脑损伤神经保护的作用及机制

The hypoxic-ischemic brain damage (HIBD) is an important cause of neonatal death and disability. While, the specific pathogenesis of HIBD has not been clarified yet, and presently a lack of effective intervention drugs. . In our privious study, we found that the expression of ubiquilin-1 (UBQLN1), a neural stem cell regulating factor, was decreased in the HIBD model of neonatal mice. Further studies showed that icarin could up-regulate UBQLN1, reduce the size of the ischemic lesion, and improve the motor function of the mice. Therefore, we hypothesized that icariin may alleviate brain injury through regulating the proliferation and differentiation of neural stem cells mediated by UBQLN1. The methods such as molecular biology, genetic engineering mice, bioinformatics are used in present study to detect the expression characteristics of UBQLN1 in HIBD model in vivo and neural stem cells in vitro, investigate the specific mechanism of icariin on regulating the proliferation and differentiation of HIBD neural stem cells in neonatal mice mediated by UBQLN1, screen out the key molecules regulating nerve regeneration by UBQLN1, and evaluate the effect of the interaction between UBQLN1 and target genes on nerve regeneration following HIBD. . This project is expected to reveal the mechanism of the protective effect of icariin on HIBD in neonatal mice and explore new strategies for prevention and treatment of neonatal HIBD.

缺氧缺血性脑损伤（HIBD）是新生儿致死、致残的重要原因，其具体发病机制尚未阐明，且缺乏有效干预药物。前期，我们在新生小鼠HIBD模型中发现神经干细胞调控因子Ubiquilin-1（UBQLN1）表达降低，进一步研究证实，淫羊藿苷可上调UBQLN1，减小缺血灶的体积并改善小鼠运动功能。据此我们提出假设：淫羊藿苷是否通过UBQLN1介导神经干细胞增殖和分化减轻HIBD。本项目拟采用分子生物学、基因工程小鼠、生物信息学等方法检测UBQLN1在HIBD模型小鼠和体外神经干细胞中的表达特征；探讨淫羊藿苷对UBQLN1调节新生鼠HIBD神经干细胞增殖、分化的具体机制；筛选出UBQLN1调节神经细胞再生的关键分子；评价UBQLN1与靶基因间的相互作用对HIBD后神经再生的效应。该项目有望揭示淫羊藿苷对新生鼠HIBD神经保护作用的机制，探索防治新生儿HIBD的新策略。

新生儿缺氧缺血性脑损伤（Hypoxic-ischemic brain damage, HIBD）是导致婴幼儿癫痫、智力残疾和脑瘫等永久性神经功能障碍疾病和急性死亡的主要原因之一。亚低温治疗（Therapeutic hypothermia, TH）作为新生儿HIBD唯一有效的治疗方法，只能适度改善预后，开发新的、更有效的治疗新生儿HIBD的方法已成为当务之急。本报告体内实验构建新生鼠HIBD模型，体外实验构建HT22细胞糖氧剥夺（Oxygen and glucose deprivation, OGD）模型。分别从体内体外检测淫羊藿苷（Icariin, ICA）对自噬相关蛋白以及Ubiquilin-1、ERα、ERβ表达的影响。ICA预处理的同时分别给予HIBD新生鼠和OGD损伤的HT22细胞自噬抑制剂3-MA、ERα抑制剂MPP、ERβ抑制剂PHTPP处理，进一步检测ICA是否可能通过激活ERα和ERβ途径促进细胞自噬进而减少细胞凋亡对新生小鼠HIBD发挥神经保护作用。ICA预处理的同时敲减Ubiquilin-1，更深入的探索Ubiquilin-1敲减对ERα、ERβ以及HIBD新生小鼠自噬凋亡、神经干细胞增殖与分化水平的影响。结果显示，ICA预处理显著促进HIBD幼鼠的细胞自噬水平。3-MA显著抑制ICA诱导的自噬水平升高，逆转了ICA的神经保护作用，促进细胞凋亡。此外，ICA预处理显著上调HIBD新生小鼠Ubiquilin-1、ERα、ERβ蛋白表达水平。MPP和PHTPP对ICA激活的ERα、ERβ蛋白表达水平升高均表现出明显的抑制作用，逆转了ICA的神经保护作用，抑制自噬，促进细胞凋亡。敲减Ubiquilin-1逆转了ICA的神经保护作用，抑制ERα、ERβ蛋白表达水平，抑制自噬，促进细胞凋亡，抑制神经干细胞增殖与分化。本研究结果表明，ICA可能通过激活Ubiquilin-1/ERα/ERβ途径促进细胞自噬进而减少HIBD所诱发的细胞凋亡，并促进神经干细胞增殖与分化，对新生鼠HIBD发挥神经保护作用。本研究揭示ICA对新生小鼠HIBD神经保护作用的新机制，为防治新生儿HIBD提供新策略。