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

项目摘要/摘要 年龄相关性特发性肺纤维化(IPF)和石棉肺的发病机制复杂， 尽管越来越多的证据确凿地表明线粒体DNA(MtDNA)受损，但人们对此还不完全了解 这导致肺泡上皮细胞(AEC)凋亡成为疾病发展过程中的关键事件。我们之前 显示AEC线粒体DNA损伤、线粒体乌头酸酶(ACO-2)耗竭和细胞凋亡的程度 石棉致纤维化潜力的关键决定因素。Sirtuin 3(SIRT3)是主要的线粒体 脱乙酰酶，并被认为是“线粒体基因组的守护神”，因为它在调节 线粒体蛋白质通过调节线粒体8-DNA来解毒氧化应激并保护线粒体DNA。 氧鸟嘌呤DNA糖基酶(OGG1)和ACO-2活性。我们为此做准备的初步研究 建议显示氧化应激(石棉或过氧化氢)降低AEC SIRT3蛋白的表达；SIRT3 沉默增强而SIRT3强制表达(EE)减弱氧化剂诱导的AEC ACO-2耗竭， 线粒体DNA损伤和细胞凋亡；SIRT3缺乏加强石棉和博莱霉素诱导的肺损伤 纤维化与血管内皮细胞线粒体DNA损伤增加相关；特发性肺疾病患者的肺 肺纤维化(IPF)增加了OGG1和MnSOD的乙酰化。综合来看，这些数据 提示SIRT3在维持健康的肺泡上皮和预防纤维化中的新作用 肺部疾病。我们假设和厚朴酚，一种小分子SIRT3诱导剂，减弱了氧化剂诱导的 AEC线粒体DNA损伤、线粒体ROS产生、细胞凋亡以及肺纤维化 维持OGG1、ACO-2和MnSOD的表达和活性。我们制定了两个相关的目标，以 检验这一假设。在目标1中，我们将确定和厚朴酚是否能阻止氧化剂诱导的aEC线粒体dna。 SIRT3依赖的AEC保存机制在体外损伤和内源性凋亡中的作用 线粒体蛋白(OGG1、ACO-2、MnSOD)的功能与蛋白质乙酰化和线粒体的减少 罗斯。在目标2中，我们将使用野生型和SIRT3-/-小鼠来确定和厚朴酚和白藜芦醇(另一种 小分子sirtuin诱导剂)通过SIRT3-减轻体内肺纤维化(石棉或博莱霉素) 肺泡2型上皮细胞的依赖机制及其保护作用是否与减少有关 (AT2)OGG1和MnSOD乙酰化、线粒体DNA损伤和细胞凋亡。这些研究将阐明 和厚朴酚诱导的SIRT3-EE在保护AT2细胞线粒体DNA完整性和预防肺纤维化中的作用 这可能对IPF和其他纤维化肺疾病的治疗具有广泛的意义。

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.