甲烷靶向TET3-OGT复合体调控Nrf2信号治疗脊髓缺血再灌注损伤的机制研究

Oxidative stress is the important pathogenic factor for spinal cord ischemia-reperfusion (IR) injury. We found that methane promoted the transcription factor Nrf2 gene and protein expressions, inhibited the negative regulatory factor Keap1 gene and protein levels, and increased Nrf2 transcription activity, which can mobilize the antioxidant defense system to ameliorate spinal cord IR injury. This work has been published on Journal of Free Radical Biology and Medicine (FRBM). However, the target of methane and its regulatory mechanism are not clear. Our latest work showed that methane upregulated and activated methyl cytosine dioxygenase TET3 which can recruit O-GlcNAc glycosyltransferase (OGT) to the gene promoter region, and contributed to formation of TET3-OGT complex. The complex can increase DNA demethylation, and promoted O-GlcNAc glycosylation of H3K4 methyltrasferase, transcriptional repressor complex and transcriptional coactivator, which can contribute to gene expression, gene silencing and increase of transcription factor activity. This project intends to use co-immunoprecipitation and other techniques, and investigates the regulatory mechanism of Nrf2 signaling through methane targeting TET3-OGT complex for treatment of spinal cord IR injury at the animal, cellular and molecular levels. This study can provide the theoretical basis for the bioactivity and medical value of methane, and identify the new drug targets for prevention and treatment of IR injury in the nervous system.

氧化应激是脊髓缺血再灌注（IR）损伤的重要致病因素。我们发现甲烷能促进抗氧化转录因子Nrf2基因转录与蛋白合成，同时抑制其负性调节因子Keap1基因和蛋白水平，并增强Nrf2转录活性，以调动机体抗氧化防御，减轻脊髓IR损伤（已发表在FRBM）。然而甲烷的作用靶点及调控机制不甚清楚。我们最新工作表明甲烷能够靶向激活甲基胞嘧啶双加氧酶TET3，并招募O-GlcNAc糖基转移酶（OGT）至基因启动子区与之形成TET3-OGT复合体。复合体既能促进DNA去甲基化，又可O-GlcNAc糖基化修饰、活化H3K4甲基转移酶、转录阻遏复合物和转录辅激活因子，以调控基因表达、沉默和转录因子活性。本项目拟采用免疫共沉淀等技术从动物、细胞和分子水平系统阐述甲烷靶向TET3-OGT复合体调控Nrf2信号治疗脊髓IR损伤的机制，为阐明甲烷的生物活性与医用价值奠定理论基础，更为防治神经系统IR损伤确立新的药物靶点。

脊髓缺血再灌注损伤是胸腹主动脉瘤等手术后严重并发症，影响患者术后康复。我们研究发现，脊髓缺血再灌注损伤大鼠细胞氧化型GSH（GSSG），超氧阴离子，丙二醛（MDA）等氧自由基（ROS）升高，IL-1β、IL-18等炎症反应标志物含量上调，提示氧化应激和炎症反应重要地参与了脊髓缺血再灌注损伤。研究发现，小分子有机物甲烷具有良好的生物安全性以及脂溶性，可以激活10-11易位蛋白3（TET3），进一步招募氧连氮-乙酰葡萄糖胺（O-GlcNAc）转移酶（OGT）至转录因子E2相关因子2（Nrf2）基因启动子区，通过OGT上调组蛋白第三亚基四号赖氨酸（H3K4）甲基转移酶复合物调节亚基RBBP5的O-GlcNAc糖基化水平，促进H3K4甲基转移酶复合物催化亚基MLL1与组蛋白H3空间邻近水平，增加Nrf2启动子区第三亚基四号赖氨酸的三甲基化组蛋白（H3K4m3）表达，诱导DNA去甲基化上调Nrf2的表达，Nrf2可以上调SOD、过氧化氢酶（CAT）基因表达及酶活性，从而清除ROS减轻氧化应激损伤。同时，甲烷可通过下调嘌呤能受体P2X配体门控离子通道7（P2X7R）转录表达，抑制钾离子外流和钙离子内流，抑制细胞内NOD样受体蛋白3（NLRP3）、凋亡相关斑点样蛋白（ASC）和半胱氨酸蛋白激酶-1（Caspase-1）组成炎症小体，下调IL-1β和IL-18，从而减轻大鼠脊髓缺血再灌注损伤时炎症反应。且甲烷下调P2X7R可能与TET3-OGT通路重要相关。课题组在地氟烷预处理减轻脂多糖（LPS）诱导大鼠脑急性炎症实验中发现神经元炎症反应与氧化应激重要相关，地氟烷能通过上调Nrf2，下调Nrf2负性调节因子Kelch样ECH相关蛋白1（Keap1），抑制氧化应激，进一步下调NLRP3、ASC、Caspase-1等表达，减少IL-18、IL-1β分泌，从而抑制炎症反应。提示在神经元氧化应激与炎症反应可能呈相互促进的正反馈级联反应。另外，临床研究也证实贫血致缺氧导致氧化应激反应，是腹腔镜手术患者术后认知功能恢复的重要影响因素。本课题旨在阐明神经元氧化应激与炎症反应的分子机制、内在关系以及甲烷优越的生物医学效应对于神经元氧化应激与炎症相关疾病防治的临床意义和巨大的开发前景。