Quercetin prevents progression of COPD lung disease by modulating Foxo3Aactivity

项目概述槲皮素是一种植物类黄酮，是一种有效的抗氧化剂和抗炎剂。我们进行了临床前研究，评估了槲皮素在慢性阻塞性肺疾病（COPD）治疗中的适用性，慢性阻塞性肺疾病是美国第三大死亡原因。我们发现槲皮素可以减少肺部炎症、杯状细胞化生和肺气肿的进展，部分原因是槲皮素增加了弹性酶/ lps暴露小鼠中脱乙酰酶Sirt1的表达，表现出COPD的典型特征。我们的初步研究表明，暴露于弹性蛋白酶/脂多糖的小鼠显示出核FOXO3a的减少，FOXO3a是一种负调节炎症并提供抗氧化应激能力的转录因子。此外，我们发现维持COPD气道上皮表型特征的COPD气道上皮细胞也显示Sirt1和FOXO3a的核水平降低。人鼻病毒（RV）感染是COPD加重的常见原因，进一步降低了核Sirt1和FOXO3a水平。我们的初步研究还表明，槲皮素促进弹性酶/脂多糖处理小鼠FOXO3a向细胞核的易位。此外，槲皮素处理COPD上皮细胞培养物可降低IL-8表达和粘液化生，同时恢复细胞核FOXO3a水平。最后，槲皮素改善了弹性酶/LPS小鼠和COPD上皮细胞在RV攻击后的病毒清除。总体目标是阐明槲皮素调节核FOXO3a和Sirt1水平的机制，从而减少慢性阻塞性肺病模型中的炎症、杯状细胞化生和增强病毒清除。为实现这一目标，我们提出以下具体目标：确定槲皮素在COPD模型中调节FOXO3a活性、抑制IL-8等趋化因子过表达、减轻肺部炎症的机制。FOXO3a的核易位受磷酸化和乙酰化调控。我们验证了以下假设：1)槲皮素治疗阻断Akt激酶活性，从而增加FOXO3A向细胞核的易位；2)槲皮素诱导的Sirt1使细胞核FOXO3a去乙酰化，阻止Akt从细胞核输出磷酸化；3) Sirt1和FOXO3a共同阻断NF-κB与cxcl - 1,2,5和8启动子的结合，从而减弱细胞因子的表达和肺部炎症。2. 研究槲皮素减少慢性阻塞性肺病气道杯状细胞化生的机制。我们的初步数据表明，在COPD细胞和COPD小鼠模型中，EGFR异常激活。我们将验证以下假设：1)槲皮素诱导的细胞核FOXO3a正调控E-cadherin的表达，E-cadherin将EGFR隔离在上皮细胞基底外表面；2)槲皮素直接抑制EGFR活性；3)槲皮素通过恢复Sirt1水平，抑制基质金属蛋白酶（MMP）的表达，从而降低EGF配体的可用性；4) EGFR活性降低降低粘蛋白基因的表达，阻止杯状细胞化生。3. 确定槲皮素增加COPD气道病毒清除率的机制。我们将验证以下假设：1)氧化应激诱导自噬体的形成和积累，为病毒复制提供平台；2) RV感染通过对Akt和CBP/p300的影响，降低核FOXO3a和II期抗氧化酶的表达，放大COPD细胞中存在的紊乱；3)槲皮素通过调节FOXO3a和Sirt1恢复抗氧化酶的表达，减少自噬，增加病毒清除率。这些研究的完成将为槲皮素减少慢性阻塞性肺疾病气道炎症、杯状细胞化生和病毒清除的机制提供重要的见解。这些研究需要确定槲皮素作为治疗慢性阻塞性肺病的替代补充药物的适用性。

DESCRIPTION (provided by applicant): Project Summary Quercetin, a plant flavonoid is a potent antioxidant and anti-inflammatory agent. We have performed preclinical studies assessing the suitability of quercetin in the treatment of chronic obstructive pulmonary disease (COPD), a third leading cause of death in the U.S. We showed that quercetin reduces lung inflammation, goblet cell metaplasia and progression of emphysema, in part by increasing expression of the deacetylase Sirt1 in elastase/LPS-exposed mice displaying typical features of COPD. Our pilot studies indicate that elastase/LPS-exposed mice show reduced nuclear FOXO3a, a transcription factor that negatively regulates inflammation and provide resistance to oxidative stress. Further, we found that COPD airway epithelial cells which maintain the phenotypic characteristics of the COPD airway epithelium also show reduced nuclear levels of Sirt1 as well as FOXO3a. Infection with human rhinovirus (RV), a common cause of COPD exacerbations further reduced nuclear Sirt1 and FOXO3a levels. Our pilot studies also show that quercetin promotes translocation of FOXO3a to the nucleus in elastase/LPS-treated mice. Further, quercetin treatment of COPD epithelial cell cultures reduced IL-8 expression and mucus metaplasia, while restoring nuclear FOXO3a levels. Finally, quercetin improved viral clearance after RV challenge in both elastase/LPS mice and COPD epithelial cells. The overall goal is to elucidate the mechanisms by which quercetin modulates nuclear FOXO3a and Sirt1 levels, thereby decreasing inflammation, goblet cell metaplasia and augment viral clearance in COPD models. To accomplish this goal, we propose the following specific Aims. 1. Determine the mechanisms by which quercetin modulates FOXO3a activity and inhibits overexpression of IL-8 and other chemokines and reduces lung inflammation in COPD models. Nuclear translocation of FOXO3a is regulated by phosphorylation and acetylation. We test the hypotheses that 1) quercetin treatment blocks Akt kinase activity, thereby increasing FOXO3A translocation to the nucleus; 2) quercetin-induced Sirt1 deacetylates nuclear FOXO3a, preventing phosphorylation by Akt export from the nucleus; and 3) Sirt1 and FOXO3a together block NF-κB binding to the CXCL-1, 2, 5 and 8 promoter, thereby attenuating cytokine expression and lung inflammation. 2. Examine the mechanisms by which quercetin decreases goblet cell metaplasia in COPD airways. Our pilot data indicate aberrant activation of EGFR in COPD cells as well as in COPD mouse model. We will test the hypotheses that 1) quercetin-induced nuclear FOXO3a positively regulates expression of E-cadherin, which sequesters EGFR at the epithelial cell basolateral surface; 2) quercetin directly inhibits EGFR activity; 3) quercetin, by restoring Sirt1 levels, inhibits matrix metalloproteinase (MMP) expression, thereby decreasing the availability of EGF ligands; and 4) reduced EGFR activity decreases expression of mucin genes, preventing goblet cell metaplasia. 3. Determine the mechanisms by which quercetin increases viral clearance in COPD airways. COPD exacerbations are associated with increased oxidative stress We will test the hypotheses that: 1) oxidative stress induces formation and accumulation of autophagosomes, providing a platform for viral replication; 2) RV infection, via its effects on Akt and CBP/p300, decreases nuclear FOXO3a and phase II antioxidant enzyme expression, amplifying derangements present in COPD cells; and 3) quercetin restores antioxidant enzyme expression by modulation of FOXO3a and Sirt1, decreasing autophagy and increasing viral clearance. Completion of these studies will provide important insights into mechanisms by which quercetin decreases airway inflammation, goblet cell metaplasia and viral clearance in chronic obstructive lung disease. These studies are required to determine the suitability of quercetin as an alternative complimentary medicine in the management of COPD lung disease.