基于NAD(+)介导的线粒体-细胞核作用平衡研究HO-1在内毒素急性肺损伤保护作用中的机制

Mitochondrial dysfunction is one of the important mechanisms involved in endotoxin induced acute lung injury (ALI). Our previous studies have shown that HO-1 could attenuate endotoxin induced ALI by regulation mitochondrial dynamics equilibrium, but the research of single organelles has not the potential to explain all phenomena at the cellular level. Recently, it has been suggested that mitonuclear communication is essential for maintaining cellular homeostasis, and the NAD(+) has emerged as a vital factor in this process beyond its role as a coenzyme. According to preliminary experiment, we found that upregulation of HO-1 could reduce the endotoxin induced ALI and decrease the mortality rate by increasing the content of NAD(+) in lung tissue. Based on the above research, we draw the hypothesis of “mitonuclear balance”, and put forward the hypothesis that HO-1 may regulate the lung tissue NAD(+) metabolism to maintain “mitonuclear balance”, and delay or reduce endotoxin induced ALI.We propose to explore the related mechanism of HO-1 in transcriptional regulation of NAD(+) in maintaining mitonuclear balance by gene knockout etc., both in vivo and in vitro.These results will have great theoretical value in clarifying the pathogenesis of endotoxin induced ALI and might provide us with new treatment strategy.

线粒体功能障碍是参与内毒素急性肺损伤（ALI）的重要机制之一。本课题组前期研究证实：HO-1可通过调控线粒体动力学平衡减轻内毒素ALI，但针对单一细胞器的研究不能很好解释发生在细胞水平的所有现象。近年来有研究认为线粒体-细胞核通信参与维持机体内稳态，且作为机体必需辅酶的NAD(+)是参与线粒体-细胞核通信的重要因子。本课题组预实验发现上调 HO-1通过增加肺组织NAD(+)含量减轻小鼠内毒素ALI并降低死亡率。基于以上研究，我们提出“线粒体-细胞核作用平衡”假说，并假设：HO-1可能通过调节肺组织细胞NAD(+)生物代谢过程维持“线粒体-细胞核作用平衡”，延迟或减轻内毒素ALI。我们拟通过基因敲除等技术，从在体和离体不同角度探索HO-1对NAD(+)的转录调控在维持“线粒体-细胞核作用平衡”中的相关机制，对进一步明确内毒素ALI的发生机制有重要理论价值，并为治疗内毒素ALI提供新思路。

内毒素急性肺损伤（ALI）是临床常见急危重症，如何有效预防和防治内毒素ALI一直是医学界研究的热点和难点课题。在国家自然科学基金（81772106）资助下，本项目已成功构建HO-1、Sirt3、NMNAT1基因敲除型小鼠，采用尾静脉注射LPS15mg/kg作用12h建立内毒素ALI模型。同时，采用共聚焦技术证实不同肺组织细胞类型中肺泡II型上皮细胞与Sirt3共定位表达，并成功培养传代肺泡II型上皮细胞，采用基因沉默方法使Sirt3、FOXO1表达降低，同时给予白藜芦醇（Sirt3诱导剂）预处理使Sirt3过表达，给予LPS10μg/ml孵育24h建立内毒素攻击肺泡上皮细胞模型。通过在体小鼠和离体肺组织细胞实验本项目观察了内毒素ALI时NAD+含量的变化及其在“线粒体-细胞核作用平衡”中的枢纽作用并初步筛选出发挥作用的NMNAT1亚型及Sirt3亚型；分别阐明基因敲除NMNAT1及Sirt3对内毒素ALI时“线粒体-细胞核作用平衡”的影响；此外，结合机体内源性保护机制，阐明不同表达水平HO-1可通过对NAD+的转录调控以维持肺组织“线粒体-细胞核作用平衡”，以减轻小鼠内毒素ALI，并初步筛选出其上游的信号通路机制。据此得出主要结论：内毒素ALI时发生线粒体-细胞核作用失衡，表现为线粒体、细胞核NAD+含量显著增加，NMNAT1mRNA表达水平升高，线粒体及细胞核形态异常，功能指标如细胞色素C氧化酶活性、MMP、ROS、RCR等异常，细胞核定位的Sirt1、Sirt6、Sirt7蛋白表达水平下调，线粒体定位的Sirt3蛋白表达水平下调，而HO-1诱导表达可进一步增加肺组织/细胞NAD+含量，改善线粒体及细胞核形态及功能指标变化，降低氧化应激指标水平，减轻内毒素ALI；且PI3K、Nrf2蛋白和HO-1蛋白表达一致性提示PI3K/Akt/Nrf2信号通路介导HO-1对NAD+表达的调控，以维持线粒体-细胞核作用平衡，为临床内毒素ALI的防治提供重要理论依据。