LC-MS/MS研究过氧化氢酶模拟物调控HIF介导的脑神经保护作用及其机制

In order to obtain small molecular probes which have good protection against oxidative stress injury to be acted as potential drugs for prevention and treatment of neurodegenerative disease，our research will focus on the regulation of HIF mediated neuroprotection by catalase mimics and its mechanism using LC-MS/MS: (a) A series of novel catalase mimics based on endogenous physiological activity of catalase will be designed, synthesized and characterized by 1HNMR、ESI-MS and IR. The catalytic activity of catalase mimics in vitro will be studied by the method of oxygen production and oxygen consumption of cell. (b) The neuroprotective effects of catalase mimetics on oxidative stress injury of H2O2 induced PC12 cells and rat hippocampal neuronal cells and its mechanism will be researched using LC-MS/MS technology. (c) The neuroprotective effects of the recombinant proteins of catalase mimics and apo-neuroglobin on oxidative stress injury of H2O2 induced PC12 cells and rat hippocampal neuronal cells and its mechanism will be investigated using LC-MS/MS and protein recombinant technology. (d) The effects of catalase mimics on oxidative stress pathway, HIF-1a and its mediated signaling pathways of nerve cells in the ischemia-reperfusion injury model of rats using LC-MS/MS and immunohistochemistry will be studied to explore the mechanism of brain protection of catalase mimetics.

为获得对氧化应激损伤具有良好保护作用的化学小分子探针，作为预防和治疗脑神经变性疾病的潜小分子药物，本项目将围绕“LC-MS/MS研究过氧化氢酶模拟物调控HIF介导的脑神经保护作用及其机制”进行如下研究：（a）基于内源性生理活性过氧化氢酶模拟物的功能性小分子设计、合成和体外过氧化氢酶活性研究。（b）运用LC-MS/MS技术研究过氧化氢酶模拟物对H2O2诱导PC12细胞和大鼠海马神经元细胞氧化应激损伤的神经保护作用及其机制。（c）运用LC-MS/MS和蛋白重组技术研究过氧化氢酶模拟物与脱辅基神经红蛋白构建的人工重组蛋白对PC12和大鼠海马神经元细胞氧化应激损伤的神经保护作用及其机制。（d）运用LC-MS/MS和免疫组化技术研究过氧化氢酶模拟物对大鼠缺血再灌注脑损伤后神经细胞的氧化应激通路、HIF-1a及其介导的信号通路的干预作用，进一步探讨过氧化氢酶模拟物发挥脑保护作用的机制。

大脑缺氧缺血损伤与氧化应激、线粒体损伤和炎症反应密切相关，抑制氧化应激和炎症反应对神经保护和治疗神经系统疾病有着重要的意义。本项目围绕“LC-MS/MS研究过氧化氢酶模拟物调控HIF介导的脑神经保护作用及其机制”按照研究计划执行，取得了有新意的研究成果，主要成果如下：（a）设计合成了一系列以三氮环、四氮环、含吡啶四氮环和卟啉环及其衍生物为配体的锰配合物，研究了它们的过氧化氢酶活性。（b）利用蛋白重组技术设计构建了含有蛋白质转导域的神经红蛋白（TAT PTD-Ngb）原核表达质粒，经低温诱导表达分离纯化得到了TAT PTD-Ngb蛋白，运用酸丙酮法制备了脱辅基蛋白并与锰卟啉重组得到了过氧化氢酶模拟物与脱辅基神经红蛋白构建的人工重组蛋白Mn-TAT PTD-Ngb。（c）运用LC-MS/MS、激光共聚焦和WB等技术研究Mn-TAT PTD-Ngb对PC12细胞氧化应激损伤的神经保护作用及其机制。（d）利用LPS诱导BV2细胞炎症反应，LPS+ATP诱导BV2细胞炎症小体的激活，运用激光共聚焦和WB等技术探究了Mn-TAT PTD-Ngb对LPS/LPS+ATP诱导的炎症反应和炎症小体激活的影响及其机制。（e）利用Aβ40诱导PC12细胞建立AD模型并初步探索了Mn-TAT PTD-Ngb对Aβ40诱导的PC12细胞损伤的保护作用。（f）运用LC-MS/MS研究了脯氨酸羟化酶抑制剂促进BMSCs增殖促进骨折愈合的分子机制。综上所述，Mn-TAT PTD-Ngb适用于治疗神经系统氧化应激损伤和炎症，可能成为治疗神经系统疾病的潜在药物。