HMGB1调节CPEB1-NR2A通路在新生儿缺氧缺血性脑损伤发病机制中的作用研究

Mechanism of hypoxic-ischemic brain damage (HIBD) is complicated without specific treatment, causing long-term neurological deficits, even death. To clarify key signal pathways in the pathogenesis of HIBD is necessary in developing specific treatments. It was reported that post-asphyxia immuno-inflammation plays an important role in the process of HIBD, but the detailed mechanisms are unclear. Previous study of our team found that: (1) High mobility group box 1 (HMGB1) is a key inflammatory factor related to HIBD-induced organ damage. HMGB1 is blood brain barrier permeable. (2) The action target of HMGB1 is NR2A subunit of NMDA receptor on the membrane of hippocampal neuron. (3) Cytoplasmic polyadenylation element binding protein 1 (CPEB1) is probably the upper stream protein in the regulation of NR2A by HMGB1. Hence we hypothesize that the synapse plasticity of hippocampal neuron is regulated by a inflammatory factor HMGB1 via CPEB1-NR2A pathway, causing HIBD, showing learning and memory disability. The study contents of this project include: (1) In vivo: HIBD rat model will be used to assess the effect of HMGB1 to the expression of CPEB1 and NR2A by RT-PCR, western blot, immunohistochemical staining, 2-DE etc, and to the ability of learning and memory by Morris water maze. CPEB1 gene silent technique will be used for reverse validation. (2) In vitro: Primary hippocampal neuron culture of neonatal SD rat and its oxygen-glucose deprivation model will be used to assess the effect of HMGB1 to the expression of CPEB1 and NR2A, and the anatomical structural changes of neuron synapses will be observed under confocal microscope or electron microscope. Plasmid transfection technique will be used for the positive validation of the regulation of CPEB1 to NR2A expression. CPEB1 gene silent technique will also be used for the reverse validation. (3) Effect of HMGB1-TLR4/RAGE signal pathway to the expression of CPEB1 will be assessed by Qrt-PCR, Western blot techniques etc. Regulation of CPEB1 expression by TLR4/RAGE signal pathway will be validated by constructing CPEB1 gene cell model vectored by Primary hippocampal neuron culture. Results in the study are beneficial for exploring novel HIBD therapeutic direction regarding the inflammatory factor HMGB1.

HIBD发病机制复杂，治疗缺乏特异性。研究显示炎症反应在HIBD中起重要作用，但具体途径不详。项目组前期研究发现：炎症因子HMGB1是HIBD时脏器受损的关键因子，可透过血脑屏障，其作用靶点是海马神经元NR2A受体，CPEB1蛋白可能是 HMGB1调节NR2A表达的主要上游分子。基于以上发现，本项目提出以下假说：HMGB1通过CPEB1-NR2A通路调节了海马神经元突触可塑性，导致HIBD，出现学习记忆能力受损。研究内容：应用HIBD大鼠模型和原代培养大鼠海马神经元缺氧无糖模型，通过Western blot、免疫组化、Morris水迷宫、质粒转染和基因沉默技术、构建CPEB1基因细胞模型等技术，从体内和体外两方面检测HMGB1对CPEB1、NR2A表达的影响，以及HMGB1-TLR4/RAGE信号通路对CPEB1表达的影响。为开发针对HMGB1的新生儿HIBD临床防治提供新思路。

神经炎症反应在缺氧缺血性脑损伤（HIBD）发病机制中的作用越来越受到重视，但具体作用途径不详。.主要研究内容是：（1）探讨早期抑制棒状受体4（TLR4）对新生大鼠缺氧缺血（HI）损伤后脑海马的保护作用；（2）探讨早期抑制TLR4对新生大鼠缺氧缺血损伤后脑海马神经免疫调节影响；（3）探讨早期抑制TLR4激活对HI损伤后脑海马神经元可塑性的影响；（4）探讨HMGB1介导M1/M2小胶质细胞极化失衡在新生大鼠HIBD的作用及机制。.主要发现是：（1）TAK-242早期处理能够通过抑制HI后TLR4信号通道激活对新生大鼠HIBD后脑海马近期及远期损伤有保护作用；（2）早期抑制TLR4通过影响HIBD后急慢性期小胶质细胞，星形胶质细胞，中性粒细胞粒细胞，NK细胞，T淋巴细胞激活及ICAM-1，补体C3a，炎症因子TNF-a，IL-1b，IL-10表达改善脑海马神经免疫紊乱；（3）早期抑制TLR4可通过减轻HIBD后脑海马PSD95，Aggrecan，NMDA受体亚单位，突触可塑性相关蛋白BDNF，CREB，pCREB表达下降及神经元树突棘密度下降影响海马神经元可塑性改变；（4）HI后新生大鼠血清及脑皮质HMGB1的表达皆明显升高，且HMGB1在外周血升高的时间点早于脑皮质；新生大鼠脑皮质HMGB1与小胶质细胞的共表达增强，且HI后72h，新生大鼠脑皮质小胶质细胞被激活；（5）HI后新生大鼠脑皮质中M1、M2型小胶质细胞的表达和RAGE皆明显增强，GL预处理可减弱M1型小胶质细胞和增强M2型小胶质细胞的表达，并可有效降低新生大鼠HI后脑梗死面积及脑水肿程度和RAGE的表达；（6）OGD条件下，HMGB1可加重小胶质细胞的神经毒性，进一步引起神经元活性降低、突触缩短，同时HMGB1基因增强可促进小胶质细胞M1型细胞因子的表达，HMGB1基因沉默可促进M2型细胞因子的表达；（7）OGD条件下，小胶质细胞RAGE-PI3K/Akt通路被激活，抑制HMGB1的表达可部分抑制RAGE-PI3K/Akt通路的激活，并降低M1型功能性细胞因子的表达和促进M2型功能性细胞因子的表达。.本项目共发表了3篇SCI科研论著（1篇为JCR Q1区期刊、2篇为JCR Q2区期刊，影响因子合计10.816）和1篇中文核心期刊论著，已培养毕业2名博士研究生及1名硕士研究生。