Nrf2-ARE信号通路在氢气干预新生儿坏死性小肠结肠炎中的机制研究

Necrotizing enterocolitis (NEC) is a serious cause of morbidity and mortality in newborn infants, with no effective prevention and treatment strategies. Despite improvements in neonatal care, the incidence of NEC has increased and its mortality remains high. Oxidative stress and inflammation are considered to be the key basis for the pathogenesis of necrotizing enterocolitis. Recently, hydrogen has been reported exerting anti-oxidative and anti-inflammatory effects by selectively reducing hydroxyl radiacls and peroxynitrites. In our previous study, we have established a reliable NEC model in newborn SD rats, and found that hydrogen-rich saline treatment could reduce the mortality and attenuate the severity of NEC. In addition, hydrogen could increase the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), one key transcription factor involved in regulating cytoprotective genes. However, its exact regulatory mechanism is still unproven. The aim of this study is to test the hypothesis that Nrf2-ARE signaling pathway plays an important role in the intervention of hydrogen in NEC. We will measure the effects of hydrogen on oxidative metabolites, on the expression, stabilization and nuclear translocation of Nrf2, and on the expression of downstream antioxidant genes in vitro and in vivo. Additionally, we will determine the hydrogen gas levels in blood, and seek the best intervention program via different administration pathways. As a result, the current study would provide a new preventive approach and potential targets for the prevention of NEC.

新生儿坏死性小肠结肠炎（NEC）发病率及死亡率较高，尚无特异性防治手段。氧化应激和炎症反应是导致NEC发病的重要原因。研究表明氢气具有抗氧化和抗炎等作用。本课题组在前期研究中：1）建立了稳定的新生大鼠NEC模型；2）发现氢气可显著降低NEC大鼠的疾病严重程度和死亡率；3）氢气可明显提高肠上皮细胞中内源性抗氧化系统关键转录因子Nrf2的表达，但具体机制不清楚。本课题拟通过肠上皮细胞模型和新生大鼠NEC模型，利用Western Blot、EMSA、RNAi等研究方法，观察氢气对氧化代谢产物的影响，检测Nrf2基因在转录水平、蛋白表达、核转位活性及其下游靶基因表达的差异，阐释Nrf2-ARE信号通路在氢气干预NEC中的具体分子机制。本研究还将通过不同给药途径，检测新生大鼠血液中氢气水平，氧化应激和炎症水平的差异，探讨最佳氢气干预NEC的方案。以上研究将为临床上NEC的防治提供新的思路和干预策略。

新生儿坏死性小肠结肠炎（ NEC）发病率及死亡率较高，尚无特异性防治手段。氧化应激和炎症反应是导致 NEC 发病的重要原因。本课题组前期研究发现氢气可显著降低 NEC大鼠的疾病严重程度和死亡率且内源性抗氧化系统关键转录因子 Nrf2 的表达明显增加，但具体机制不清楚。据此，本课题首先以氢气干预Caco-2、IEC-6细胞氧化应激模型，通过分析JC-1线粒体膜电位、CCK-8细胞增殖等结果发现氢气可以显著降低氧化损伤，清除氧自由基，减少脂质过氧化物，稳定线粒体膜电位减少细胞凋亡。而Real time PCR、Western Blot等结果显示氢气能够通过下调促炎因子mRNA表达并上调Nrf2的mRNA及蛋白表达进而干预肠上皮细胞氧化应激损伤。然后通过慢病毒载体介导的沉默和过表达Keap1、免疫共沉淀等方法证实氢气是通过促进Nrf2转位、降低Keap1介导的Nrf2泛素化后激活Nrf2/ARE信号通路，从而提高机体内源性抗氧化应激损害的能力。临床病例检测结果显示，与健康早产儿相比，NEC患儿外周血Nrf2和HO-1的mRNA表达显著降低。最后，以经胃管注入氢饱和配方奶和经腹腔注射氢饱和生理盐水这两种不同给氢方式对新生大鼠NEC模型进行干预，通过对real-time PCR、western blot 和免疫组织化学染色等结果分析发现氢气摄入可以维持模型动物体重增长，降低发病率和死亡率，减轻疾病严重程度，减轻炎症因子表达、增加Nrf2等抗氧化因子表达，且经胃管内注入氢饱和配方乳上调肠道Nrf2蛋白表达、激活SOD能力更显著。综上所述，本课题研究成果成功揭示了Nrf2-ARE信号通路在氢气干预 NEC 中的具体分子机制，为临床上 NEC 的防治提供新的思路和干预策略，同时发现氢饱和配方乳为更佳的氢气干预 NEC 方案，对今后氢气的临床应用途径具有参考意义。项目资助发表SCI论文2篇，中文核心论文3篇，培养博士生1名，硕士生1名。