脑缺血早期乳酸通过NDRG2促进反应性星形胶质细胞形成的作用及机制研究

Lactate is an energy supply substance for nervous system. During cerebral ischemia, the extracellular lactate concentration within lesion area rises rapidly, and astrocytes (ACs) are activated to reactive astrocytes (RACs) to play crucial roles in preventing the spread of damage and promoting tissue repair. However, that is still unclear if the high concentration of extracellular lactate stimulates ACs differentiating into RACs phenotype. NDRG2 is one of the stress-associated genes, which is specially expressed in ACs in the central nervous system. In preliminary study, through molecular docking analysis we have found that there is a lactate binding pocket in the three-dimensional structure of NDRG2 protein. Additionally, in vitro hypoxia and high concentration of lactate culture condition, the NDRG2, pSTAT3 and pAKT protein expression of ACs were significantly upregulated. Accordingly, we hypothesize that in early phase of the ischemia damage the accumulated extracellular lactate acts as a key signaling molecular to trigger the activation of JAK/STAT3 and mTOR pathways in ACs, causing RACs phenotype. In this study, in vitro hypoxia condition and relatively higher concentration of lactate, similar with brain ischemic state, we will investigate the relationships between the extracellular lactate and the target protein NDRG2, as well as the mechanism of lactate in activating ACs differentiating into RACs. This study will explore a novel biological function of lactate during early phase of cerebral ischemia and provide avenue for discovering new therapeutic strategy for cerebral ischemia.

乳酸是神经系统能量提供物质之一，脑缺血后损伤部位细胞外乳酸含量急剧增高。损伤早期星形胶质细胞（ACs）活化为反应性星形胶质细胞（RACs），有保护和促进组织修复的作用。但形成RACs过程中细胞外乳酸的调控作用一直未受到关注。NDRG2是一种应激反应基因，在中枢神经系统中特异表达于ACs。申请人通过分子对接分析发现NDRG2蛋白立体结构中有乳酸分子结合口袋，在缺氧并富含乳酸的培养条件下，ACs高表达NDRG2、pSTAT3和pAKT蛋白，活化为RACs。据此推测：损伤早期细胞外乳酸可能作为一种关键信号分子激活JAK/STAT3和mTOR通路，活化ACs 为RACs。本项目在体外模拟脑缺血高浓度乳酸微环境，以NDRG2为乳酸结合靶分子探究细胞外高浓度乳酸活化ACs为RACs的作用及分子机制，阐明脑缺血早期乳酸新的生物学作用，为探索治疗脑缺血损伤的新方法和策略提供实验依据。

乳酸是神经系统能量提供物质之一，脑缺血后损伤部位细胞外乳酸含量急剧增高。损伤早期星形胶质细胞（ACs）活化为反应性星形胶质细胞（RACs），有保护和促进组织修复的作用。但形成RACs过程中细胞外乳酸的调控作用一直未受到关注。NDRG2在中枢神经系统中特异表达于ACs。本项目提出了“脑缺血早期损伤部位蓄积的细胞外乳酸进入ACs与NDRG2结合促进ACs向RACs活化”的科学假说。为此项目组利用大鼠拴线阻塞脑缺血（MCAO）模型，体外缺氧缺糖（OGD）+高乳酸的ACs培养模型以及NDRG2敲除小鼠模型，应用细胞和分子生物学方法和技术，开展了以下研究：1）确定细胞外乳酸是否进入ACs极化其为RACs；2）确定细胞外乳酸是否通过MCT向RACs细胞内有效转运；3）证明乳酸和NDRG2 蛋白结合抑制NDRG2蛋白降解；4）以NDRG2为靶分子阐明乳酸是否介导NDRG2活化ACs为RACs以及其他作用；5）建立ACs的NDRG2基因敲除小鼠模型，制作MCAO观察NDRG2缺失对RACs的影响。另外，应用体外OGD小胶质细胞（MGs）培养模型，研究OGD高乳酸对MG2极化的作用以及机制。. 研究发现：1）大鼠MCAO后8h，损伤部位的乳酸浓度显著升高，ACs中NDRG2、SOX9和GFAP表达显著上调，激活AKT和STAT3通路，ACs极化为RACs发生胶质增生；2）细胞外乳酸通过MCT1进入ACs内发挥作用；4）分子对接分析发现，在NDRG2氨基酸序列中，Lys176和Arg240分别是与乳酸氢键结合最强的位点；5）其中Lys176与乳酸氢键结合有效抑制NDRG2泛素化稳定蛋白，是关键结合位点；6）通过体外细胞实验和NDRG2敲除小鼠MCAO模型，证明乳酸不是与NDRG2结合促进ACs向RACs极化，而是通过NDRG2抑制c-Jun蛋白磷酸化减少TNFa等促炎因子表达和ACs凋亡；7）通过体外实验发现OGD和OGD+高乳酸条件，促进MGs向具有抗炎作用的M2型极化；8）证明乳酸通过DAB2蛋白抑制p38 MAPK通路促进MGs向M2型极化。. 本研究，从ACs和MGs角度为重新认识“脑缺血损伤早期乳酸的神经保护作用和机制”提出了新理论和支持的实验数据。并且为以乳酸为生物活性物质、以NDRG2和DAB2为靶蛋白研究开发脑缺血损伤治疗方案，提供理论和实验依据。