The role of SIRT3 inducers in preventing alveolar epithelial cell death and lung fibrosis

项目总结/摘要 与年龄相关的特发性肺纤维化（IPF）和石棉肺的发病机制是复杂的， 尽管越来越多的证据表明线粒体DNA（mtDNA）损伤， 其导致肺泡上皮细胞（AEC）凋亡作为疾病发展中的关键事件。我们之前 结果表明，AEC线粒体DNA损伤、线粒体顺乌头酸酶（ACO-2）缺失和细胞凋亡的程度是 石棉纤维化潜力的关键决定因素。Sirtuin 3（SIRT 3）是线粒体中主要的蛋白质， 脱乙酰酶，并认为是“线粒体基因组的监护人”，通过其在调节 线粒体蛋白质，解毒氧化应激，并通过调节线粒体8- 氧代鸟嘌呤DNA糖基化酶（OGG 1）和ACO-2活性。我们的初步研究， 研究表明，氧化应激（石棉或H2 O2）降低AEC SIRT 3蛋白表达; SIRT 3 沉默增强而SIRT 3强制表达（EE）减弱氧化剂诱导的AEC ACO-2消耗， 线粒体DNA损伤和细胞凋亡; SIRT 3缺乏增强石棉和博来霉素诱导的肺 与AEC mtDNA损伤增加相关纤维化;以及特发性肺纤维化患者的肺 肺纤维化（IPF）患者的OGG 1和MnSOD的乙酰化增加。综合来看，这些数据 提示SIRT 3在维持健康肺泡上皮细胞和预防纤维化中的新作用。 肺部疾病我们推测，和诺啡肽，一种小分子SIRT 3诱导剂， AEC线粒体DNA损伤、线粒体ROS产生、细胞凋亡以及肺纤维化部分是由 维持OGG 1、ACO-2和MnSOD的表达和活性。我们制定了两个相关的目标， 测试这个假设。在目的1中，我们将确定和厚朴酚是否阻止氧化剂诱导的AEC mtDNA 体外通过SIRT 3依赖性机制保护AEC的损伤和内在凋亡 线粒体蛋白（OGG 1，ACO-2，MnSOD）功能和蛋白乙酰化和线粒体 罗斯在目标2中，我们将使用野生型和Sirt 3-/-小鼠来确定和厚朴酚和白藜芦醇（另一种 小分子沉默调节蛋白诱导剂）通过SIRT 3- 依赖性机制以及保护作用是否与肺泡上皮2型细胞减少有关 (AT2)OGG 1和MnSOD乙酰化，线粒体DNA损伤和细胞凋亡。这些研究将阐明 和厚朴酚诱导SIRT 3-EE对AT 2细胞线粒体DNA完整性和肺纤维化的保护作用 这可能对IPF和其他纤维化肺病的治疗具有广泛意义。

Project Summary/Abstract The pathogenesis of age-related idiopathic pulmonary fibrosis (IPF) and asbestosis is complex and incompletely understood, though accumulating evidence firmly implicates mitochondrial DNA (mtDNA) damage that lead to alveolar epithelial cell (AEC) apoptosis as a key event in disease development. We previously showed that the extent of AEC mtDNA damage, mitochondrial aconitase (ACO-2) depletion and apoptosis are critical determinants of the fibrogenic potential of asbestos. Sirtuin 3 (SIRT3) is the major mitochondrial deacetylase and considered the “guardian of the mitochondrial genome” through its role in regulating mitochondrial proteins that detoxify oxidative stress and preserve mtDNA via modulation of mitochondrial 8- oxoguanine DNA glycosylase (OGG1) and ACO-2 activities. Our preliminary studies in preparation for this proposal show that oxidative stress (asbestos or H2O2) decreases AEC SIRT3 protein expression; that SIRT3 silencing augments while SIRT3 enforced expression (EE) attenuates oxidant-induced AEC ACO-2 depletion, mtDNA damage, and apoptosis; that SIRT3 deficiency enhances asbestos- and bleomycin-induced pulmonary fibrosis in association with increased AEC mtDNA damage; and that lungs from patients with idiopathic pulmonary fibrosis (IPF) have increased acetylation of OGG1 and MnSOD. Taken together, these data suggest a novel role for SIRT3 in the maintenance of a healthy alveolar epithelium and prevention of fibrotic lung diseases. We hypothesize that honokiol, a small molecule SIRT3 inducer, attenuates oxidant-induced AEC mtDNA damage, mitochondrial ROS production, and apoptosis as well as pulmonary fibrosis in part by maintaining expression and activity of OGG1, ACO-2, and MnSOD. We have formulated two related aims to test this hypothesis. In Aim 1, we will determine whether honokiol prevents oxidant-induced AEC mtDNA damage and intrinsic apoptosis in vitro via a SIRT3-dependent mechanism involving preservation of AEC mitochondrial protein (OGG1, ACO-2, MnSOD) function and reduction in protein acetylation and mitochondrial ROS. In Aim 2, we will use wild-type and Sirt3-/- mice to determine whether honokiol and resveratrol (another small molecule sirtuin inducer) mitigate pulmonary fibrosis (asbestos or bleomycin) in vivo by a SIRT3- dependent mechanism and whether protection is associated with reductions in alveolar epithelial type 2 cell (AT2) OGG1 and MnSOD acetylation, mtDNA damage, and apoptosis. These studies will elucidate the importance of honokiol-induced SIRT3-EE in preserving AT2 cell mtDNA integrity and preventing lung fibrosis which may have broad implications for the treatment of IPF and other fibrotic lung diseases.